Methods and compositions for liposomal formulation of antigens and uses thereof

ABSTRACT

The present invention relates to liposomal vaccine compositions, methods for the manufacture thereof, and methods for the use thereof to stimulate an immune response in an animal. These compositions comprise dimyristoylphosphatidylcholine (“DMPC”); either dimyristoylphosphatidylglycerol (“DMPG”) or dimyristoyltrimethylammonium propane (“DMTAP”) or both DMPC and DMTAP; and at least one sterol derivative providing a covalent anchor for one or more immunogenic polypeptide(s) or carbohydrate(s).

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 12/720,592,filed Mar. 9, 2010, now U.S. Pat. No. 8,765,171, issued Jul. 1, 2014,which claims the benefit of U.S. Provisional Application No. 61/158,694,filed Mar. 9, 2009, all of which are hereby incorporated in theirentirety including all tables, figures, and claims.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of NationalInstitute of Allergy and Infectious Diseases (NIH) Grant No.1R43AI077119-01; Grant No. NIH R01 GM061851; National Institutes ofHealth, University of California, San Francisco—Gladstone Institute ofVirology & Immunology Center for AIDS Research, P30-AI027763; and U.S.Department of Homeland Security Graduate Fellowship contract numberDE-AC05-000R22750.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Liposomes are vesicles formed from one (“unilamellar”) or more(“multilamellar”) layers of phospholipid. Because of the amphipathiccharacter of the phospholipid building blocks, liposomes typicallycomprise a hydrophilic layer presenting a hydrophilic external face andenclosing a hydrophilic core. The versatility of liposomes in theincorporation of hydrophilic/hydrophobic components, their non-toxicnature, biodegradability, biocompatibility, adjuvanticity, induction ofcellular immunity, property of sustained release and prompt uptake bymacrophages, makes them attractive candidates for the delivery ofantigens.

Liposomes have been demonstrated to induce both humoral andcell-mediated immunity to a large variety of bacterial, protozoan, viraland tumour cell antigens. While the widespread use of liposomal vaccineshas been long anticipated, few such vaccines have been developedcommercially. The immunoadjuvant action of liposomes depends on variousstructural characteristics. Such characteristics include thethree-dimensional conformation of the antigen being presented by theliposome, which may not always mimic the natural conformation of theantigen.

For example, the membrane proximal region (MPR) of HIV gp41, a segmentcomprised of approximately 35 amino acids N terminal to thetransmembrane domain, has been considered a desirable vaccine targetbecause it is well conserved across viral clades and is essential forvirus-cell fusion. However, efforts to date have not succeeded ineliciting a useful immune response, and attempts to present structurallyconstrained epitopes, either conjugated to carrier proteins or graftedon recombinant constructs, have not elicited neutralizing antibodies. Inaddition to a lack of consensus regarding the epitope structure, therelatively weak immunogenicity of the MPR may result in immune responsesto recombinant envelope immunogens directed toward immunodominantregions on gp41 that mask the MPR from antibody recognition.

In addition, such characteristics may also include factors which controlvesicle fate in vivo. Methods for associating an antigen with a liposomeprior to liposome formation often expose the antigen to detergentsand/or organic solvents. In contrast, methods for associating an antigenwith a liposome following formation can expose the liposome tounfavorable chemical treatments. Liposomes may be quickly cleared by thereticuloendothelial system and macrophages, reducing the efficiency ofthe liposome as a vaccine.

There remains in the art a need for methods and compositions which canprovide liposomal vaccines that deliver antigens in a manner useful forstimulating an immune response.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide liposomal vaccinecompositions, methods for the manufacture thereof, and methods for theuse thereof to stimulate an immune response in an animal.

In one aspect, the invention relates to a composition comprising one ormore immunogenic polypeptides or carbohydrates of interest. Thecompositions of the present invention comprise:

a) an aqueous vehicle;

b) liposomes comprising

-   -   (i) dimyristoylphosphatidylcholine (“DMPC”),    -   (ii) dimyristoylphosphatidylglycerol (“DMPG”),        dimyristoyltrimethylammonium propane (“DMTAP”), or both DMPG and        DMTAP, and    -   (iii) at least one sterol derivative; and

c) one or more immunogenic polypeptide(s) or carbohydrate(s) covalentlylinked to between 1% and 100% of said at least one sterol derivative.

And in a related aspect, the invention relates to a compositioncomprising one or more immunogenic polypeptides or carbohydrates ofinterest. The compositions of the present invention comprise:

a) an aqueous vehicle; and

b) liposomes comprising

-   -   (i) dimyristoylphosphatidylcholine (“DMPC”),    -   (ii) dimyristoylphosphatidylglycerol (“DMPG”),        dimyristoyltrimethylammonium propane (“DMTAP”), or both DMPG and        DMTAP, and    -   (iii) at least one reactive sterol derivative,    -   wherein between 1% and 100% of said at least one sterol        derivatives comprise a functional moiety covalently attached        thereto, said functional moiety selected from the group        consisting of an amine reactive group, a sulfhydryl reactive        group, a carboxyl reactive group, a photoaffinity reactive        group, an arginine linking group, and a carbonyl reactive group.

For the sake of convenience, the lipid(s) selected in part (ii) abovewill be referred to below as DMPG/DMTAP, which is intended to mean DMPG,DMTAP, or a mixture of the two. In certain embodiments, the relativepercentages of DMPC, DMPG/DMTAP, and sterol derivative are 50% to 98%DMPC: 1% to 25% DMPG/DMTAP: 1% to 25% sterol derivative, and in certainother embodiments 70% to 98% DMPC: 1% to 15% DMPG/DMTAP: 1% to 15%sterol derivative. This is not meant to imply that no other componentsare present in the liposome; rather, these represent the relativepercentages of DMPC, DMPG/DMTAP, and sterol derivative on a molar basisto one another. In certain embodiments, a liposome can also contain oneor more additional components which are well known in the art, such aspeptidoglycan, lipopeptide, lipopolysaccharide, monophosphoryl lipid A,lipoteichoic acid, resiquimod, imiquimod, flagellin, oligonucleotidescontaining unmethylated CpG motifs, α-galactosylceramide, muramyldipeptide, all-trans retinoic acid, double-stranded viral RNA, heatshock proteins, dioctadecyldimethylammonium bromide, cationicsurfactants, toll-like receptor agonists,dimyristoyltrimethylammoniumpropane, and nod-like receptor agonists.

In preferred embodiments, these relative percentages are 70% to 85%DMPC: 5% to 15% DMPG/DMTAP: 10% to 15% sterol derivative, and morepreferably about 75% DMPC, about 10% DMPG/DMTAP, and about 15% sterolderivative. The term “about” as used herein in this context refers to+/−10% of a given measurement. DMPG is particularly preferred as thelipid selected in part (ii) above.

The term “sterol derivative” as used herein refers to any moleculehaving the 4-member ring structure characteristic of steroids and ahydroxyl (—OH) or ester (-OR) substitution at the 3-carbon position, andsome or all of which serves as an anchor for attaching an immunogenicpolypeptide or carbohydrate to a liposome:

The skilled artisan will understand that a sterol derivative can befurther substituted at one or more of the other ring carbons, and mayalso contain various double bonds in the rings. In certain embodiments,a sterol derivative is a derivative in which the immunogenic polypeptideor carbohydrate is covalently linked to the steroid ring structure viathe 3-carbon position, or via the 17 carbon position. Preferred sterolderivatives include derivatives of cholesterol, cholesterylchloroformate, stigmasterol, sitosterol, ergosterol, lanosterol,desmosterol, and campesterol. This list is not meant to be limiting.

In preferred embodiments, these sterol derivatives are cholesterolderivatives. A cholesterol derivative is substituted at the 8-, 10-, and13-carbon positions with methyl groups and contains a double bond at the5,6-carbon position. Most preferably, the sterol derivatives have thefollowing structure:

wherein:

-   one of R1 or R2 is a covalent linkage to an immunogenic polypeptide    or carbohydrate, wherein if R1 is said covalent linkage to said    polypeptide, R2 is H, and-   if R2 is said covalent linkage to said immunogenic polypeptide, R1    is —CH₂—CH₂—CH₂—C(H)(CH₃)₂.

In particularly preferred embodiments, R1 is —CH₂—CH₂—C(O)—X, wherein Xis an immunogenic polypeptide or carbohydrate, and R2 is H. In otherparticularly preferred embodiments, R1 is —CH₂—CH₂—CH₂—C(H)(CH₃)₂, andR2 is —C(O)—CH₂—CH₂—C(O)—X, wherein X is an immunogenic polypeptide orcarbohydrate.

In other preferred embodiments, these sterol derivatives are cholesterolderivatives. A cholesterol derivative is substituted at the 8-, 10-, and13-carbon positions with methyl groups and contains a double bond at the5,6-carbon position. Most preferably, the sterol derivatives have thefollowing structure:

wherein:

-   one of R1 or R2 is comprise a functional moiety covalently attached    thereto, wherein said functional moiety is selected from the group    consisting of an amine linking group, a sulfhydryl linking group, a    carboxyl linking group, a photoaffinity linking group, an arginine    linking group, and a carbonyl linking group, and wherein-   if R1 is said functional moiety, R2 is H, and-   if R2 is said functional moiety, R1 is —CH₂—CH₂—CH₂—C(H)(CH₃)₂.

In certain embodiments, the liposome are provided within a particularaverage size range, as size can affect the efficiency with whichliposomes are taken up when delivered mucosally, and/or cleared whendelivered intravenously. Liposome size can be determined by methods wellknown in the art, including photon correlation spectroscopy, dynamiclight scattering, etc. In preferred embodiments, the liposomes aresubstantially between 50 and 500 nm in diameter, more preferablysubstantially between 50 and 200 nm in diameter, and most preferablysubstantially between 50 and 150 nm in diameter. The term“substantially” as used herein in this context means that at least 75%,more preferably 80%, and most preferably at least 90% of the liposomesare within the designated range.

As noted above, some or all of a steroid derivative which is a componentpart of the liposome serves as an anchor for attaching an immunogenicpolypeptide or carbohydrate to a liposome. In preferred embodiments,said one or more immunogenic polypeptide(s) are covalently linked tobetween about 1% and about 25% of the sterol derivative(s), and mostpreferably between about 5% and 10% of the sterol derivative(s). Theterm “about as used herein in this context refers to +/−20% of a recitedpercentage.

As is also noted above, the liposomes of the present invention may alsocomprise one or more additional components, such as peptidoglycan,lipopeptide, lipopolysaccharide, monophosphoryl lipid A, lipoteichoicacid, resiquimod, imiquimod, flagellin, oligonucleotides containingunmethylated CpG motifs, α-galactosylceramide, muramyl dipeptide,all-trans retinoic acid, double-stranded viral RNA, heat shock proteins,dioctadecyldimethylammonium bromide,dimyristoyltrimethylammoniumpropane, cationic surfactants, toll-likereceptor agonists, and nod-like receptor agonists. These additionalcomponents can serve as additional adjuvant materials. In certainembodiments, the relative percentage of such an additional component isless than 10% of the total of DMPC, DMPG/DMTAP, and sterol derivative ona molar basis. More preferably, the relative percentage of such anadditional component is less than 2% of the total of DMPC, DMPG/DMTAP,and sterol derivative on a molar basis, and most preferably less than 1%of the total of DMPC, DMPG/DMTAP, and sterol derivative on a molarbasis.

Methods for covalently linking an immunogenic polypeptide orcarbohydrate to a sterol derivative are well known in the art. Chemicalcross-linkers are discussed in numerous books and catalogues. See, e.g.,Wong, Chemistry of Protein Conjugation and Cross-linking, CRC Press,Boca Raton, Fla., 1991. These reagents often employ functional groupsthat couple to amino acid side chains of peptides. Moieties that can betargeted using a cross-linker include primary and ε-amines, sulfhydryls,carbonyls, hydroxyls, and carboxylic acids. In addition, many reactivegroups can be coupled nonselectively using a cross-linker such asphotoreactive phenyl azides.

In the case of immunogenic polypeptide(s), these may be preferablycovalently linked to one or more sterol derivatives through one or moreof the following: a lysine residue on the immunogenic polypeptide(s),through a cysteine residue on the immunogenic polypeptide(s), through anaspartate residue on the immunogenic polypeptide(s), through a glutamateresidue on the immunogenic polypeptide(s), through a serine residue onthe immunogenic polypeptide(s), through a threonine residue on theimmunogenic polypeptide(s), through an N-terminal amine on theimmunogenic polypeptide(s), and/or through a C-terminal carboxyl on theimmunogenic polypeptide(s). In the case of immunogenic carbohydrates,these may be preferably covalently linked through a hydroxyl on theimmunogenic carbohydrate.

A covalent linkage between an immunogenic polypeptide or carbohydrateand a sterol derivative may be as short as a covalent bond between asterol ring atom or sterol side chain atom, but preferably provides oneor more linker atoms connecting the immunogenic polypeptide orcarbohydrate to the sterol derivative. Preferred linkages are C₁₋₁₈alkylene straight or branched chain comprising from 0-4 backbone (i.e.,non-substituent) heteroatoms, optionally substituted with from 1 to 4substituents independently selected from the group consisting of C₁₋₆alkyl straight or branched chain, halogen, C₁₋₆ alkoxy, —NO₂, —NH₂, ═O,—OH, —CH₂OH, trihalomethyl, —C(O)NH₂ and —C(O)(OR4) where R4 is H orC₁₋₃ alkyl.

The inclusion of polymer portions (e.g., polyethylene glycol (“PEG”)homopolymers, polypropylene glycol homopolymers, other polyalkyleneoxides, bis-polyethylene oxides and co-polymers or block co-polymers ofpoly(alkylene oxides)) in cross-linkers can, under certain circumstancesbe advantageous. See, e.g., U.S. Pat. Nos. 5,643,575, 5,672,662,5,705,153, 5,730,990, 5,902,588, and 5,932,462; Fleiner et al.,Bioconjug. Chem. 12 (4), 470-75, 2001; and Topchieva et al., Bioconjug.Chem. 6: 380-8, 1995). A preferred linkage to between an immunogenicpolypeptide or carbohydrate and a sterol derivative comprises an(alkylene oxide)_(n) moiety having an average length n of between 40 and1000. Suitable polyalkylene oxides include, but are not limited to,homopolymers and copolymers comprising methylene oxide, ethylene oxide,propylene oxide, isopropylene oxide, and butylene oxide.

In particularly preferred embodiments, a covalent linkage between animmunogenic polypeptide or carbohydrate and a sterol derivative has thestructure St-R3-X, wherein:

-   St is a ring atom of the sterol derivative;-   R3 is C₀₋₁₈ straight or branched chain alkyl, or C₀₋₁₂ straight or    branched chain alkyl-(alkylene oxide)_(n)-C₀₋₁₂ straight or branched    chain alkyl, wherein n is on average between 40 and 1000;-   each said straight or branched chain alkyl comprises from 0-4 chain    heteroatoms and one or more substituents independently selected from    the group consisting of halogen, trihalomethyl, —C₁₋₆ alkoxy, —NO₂,    —NH₂, —OH, —CH₂OH, —CONH₂, and —C(O)(OR4) where R4 is H or C₁₋₃    alkyl; and-   X is an atom of the immunogenic polypeptide or carbohydrate.

In preparing the immunogenic polypeptide-linked sterol derivatives, itis advantageous to prepare an intermediate sterol derivative in whichthe linkage chemistry terminates in a reactive group which forms acovalent bond with a reactive partner on the immunogenic polypeptide ofinterest. As discussed above, suitable reactive partners include freeamines, sulfhydryls, carboxyls, arginines, carbonyls, etc. Thus, inanother aspect, the present invention also relates to reactive sterolderivatives.

In preferred embodiments, these reactive sterol derivatives arecholesterol derivatives. A cholesterol derivative is substituted at the8-, 10-, and 13-carbon positions with methyl groups and contains adouble bond at the 5,6-carbon position. Most preferably, the sterolderivatives have the following structure:

wherein:

-   one of R1 or R2 is a covalent linkage comprising a reactive group    which reacts to form a covalent bond with a reactive partner on an    immunogenic polypeptide of interest, wherein-   if R1 is said covalent linkage to said polypeptide, R2 is H, and-   if R2 is said covalent linkage to said immunogenic polypeptide, R1    is —CH₂—CH₂—CH₂—C(H)(CH₃)₂.

In particularly preferred embodiments, R1 is —CH₂—CH₂—C(O)—RG, whereinRG is a reactive group, and R2 is H. In other particularly preferredembodiments, R1 is —CH₂—CH₂—CH₂—C(H)(CH₃)₂, and R2 is—C(O)—CH₂—CH₂—C(O)—RG, wherein RG is a reactive group. Preferredreactive groups are selected from the group consisting of imidoesters,N-hydroxysuccinimidyl (“NHS”) esters, maleimides, alkyl halides, arylhalides, α-haloacyls, pyridyl disulfides, carbodiimides, glyoxals,amines, hydrazides, and arylazides. See, e.g., Wong, Chemistry ofProtein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla.,1991.

A covalent linkage between a sterol derivative and a reactive group maybe as short as a covalent bond between a sterol ring atom or sterol sidechain atom, but preferably provides one or more linker atoms connectingthe sterol ring atom or sterol side chain atom to the reactive group.Preferred linkages are C₁₋₁₈ alkylene straight or branched chaincomprising from 0-4 backbone (i.e., non-substituent) heteroatoms,optionally substituted with from 1 to 4 substituents independentlyselected from the group consisting of C₁₋₆ alkyl straight or branchedchain, halogen, C₁₋₆ alkoxy, —NO₂, —NH₂, ═O, —OH, —CH₂OH, trihalomethyl,—C(O)NH₂ and —C(O)(OR4) where R4 is H or C₁₋₃ alkyl.

In other preferred embodiments, a covalent linkage between a sterolderivative and a reactive group has the structure St-R3-RG, wherein:

-   St is a ring atom of the sterol derivative;-   R3 is C₀₋₁₈ straight or branched chain alkyl, or C₀₋₁₂ straight or    branched chain alkyl-(alkylene oxide)_(n)-C₀₋₁₂ straight or branched    chain alkyl, wherein n is on average between 40 and 1000;-   each said straight or branched chain alkyl comprises from 0-4 chain    heteroatoms and one or more substituents independently selected from    the group consisting of halogen, trihalomethyl, —C₁₋₆ alkoxy, —NO₂,    —NH₂, —OH, —CH₂OH, —CONH₂, and —C(O)(OR4) where R4 is H or C₁₋₃    alkyl; and-   RG is a reactive group.

In another aspect, the invention relates to methods for preparingcompositions comprising one or more immunogenic polypeptides orcarbohydrates of interest. These methods comprise:

(a) covalently coupling one or more immunogenic polypeptides orcarbohydrates to one or more sterol derivatives to provide one or moreconjugated sterol derivatives; and

(b) combining

-   -   (i) dimyristoylphosphatidylcholine (“DMPC”),    -   (ii) dimyristoylphosphatidylglycerol (“DMPG”),        dimyristoyltrimethylammonium propane (“DMTAP”), or both DMPG and        DMTAP, and    -   (iii) one or more sterol derivatives, wherein between 1% and        100% of said sterol derivative(s) is(are) said conjugated sterol        derivative(s) to provide a lipid mixture; and

(c) preparing liposomes from said lipid mixture.

In a related aspect, the invention relates to methods for preparing theforegoing compositions comprising one or more immunogenic polypeptidesor carbohydrates of interest. These methods comprise:

(a) combining

-   -   (i) dimyristoylphosphatidylcholine (“DMPC”),    -   (ii) dimyristoylphosphatidylglycerol (“DMPG”),        dimyristoyltrimethylammonium propane (“DMTAP”), or both DMPG and        DMTAP, and    -   (iii) at least one sterol derivative to provide a lipid mixture;

(b) preparing liposomes from said lipid mixture; and

(c) covalently coupling one or more immunogenic polypeptides orcarbohydrates to said at least one sterol derivative, wherein said oneor more immunogenic polypeptide(s) or carbohydrate(s) are covalentlylinked to between 1% and 100% of said at least one sterol derivative.

Suitable methods for preparing liposomes from lipid mixtures are wellknown in the art. See, e.g., Basu & Basu, Liposome Methods and Protocols(Methods in Molecular Biology), Humana Press, 2002; Gregoriadis,Liposome Technology, 3^(rd) Edition, Informa HealthCare, 2006. Preferredmethods include extrusion, homogenization, and sonication methodsdescribed therein. An exemplary method for preparing liposomes of theinvention, which comprises drying a lipid mixture, followed by hydrationin an aqueous vehicle and sonication to form liposomes, is describedhereinafter. Preferred steroid derivatives, methods for covalentlycoupling immunogenic polypeptides or carbohydrates to such derivatives,and covalent linkages are discussed in detail above and hereinafter.

In certain embodiments, the relative percentages of DMPC, DMPG/DMTAP,and sterol derivative are 50% to 98% DMPC: 1% to 25% DMPG/DMTAP: 1% to25% sterol derivative, and in certain other embodiments 70% to 98% DMPC:1% to 15% DMPG/DMTAP: 1% to 15% sterol derivative. As discussed above,this is not meant to imply that no other components are present in thelipid mixture (and hence in the liposomes); rather, these represent therelative percentages of DMPC, DMPG/DMTAP, and sterol derivative on amolar basis to one another. In certain embodiments, a lipid mixture canalso contain one or more additional components which are well known inthe art, such as peptidoglycan, lipopeptide, lipopolysaccharide,monophosphoryl lipid A, lipoteichoic acid, resiquimod, imiquimod,flagellin, oligonucleotides containing unmethylated CpG motifs,α-galactosylceramide, muramyl dipeptide, all-trans retinoic acid,double-stranded viral RNA, heat shock proteins,dioctadecyldimethylammonium bromide, cationic surfactants, toll-likereceptor agonists, dimyristoyltrimethylammoniumpropane, and nod-likereceptor agonists.

In preferred embodiments, these relative percentages are 70% to 85%DMPC: 5% to 15% DMPG/DMTAP: 10% to 15% sterol derivative, and morepreferably about 75% DMPC, about 10% DMPG/DMTAP, and about 15% sterolderivative. The term “about” as used herein in this context refers to+/−10% of a given measurement. DMPG is particularly preferred as thelipid selected in part (ii) above.

In certain embodiments, methods further comprise selecting liposomeswithin a particular average size range. Liposome size can be selected,for example, by extrusion of an aqueous vehicle comprising liposomesthrough membranes having a preselected pore size and collecting thematerial flowing through the membrane. In preferred embodiments, theliposomes are selected to be substantially between 50 and 500 nm indiameter, more preferably substantially between 50 and 200 nm indiameter, and most preferably substantially between 50 and 150 nm indiameter. The term “substantially” as used herein in this context meansthat at least 75%, more preferably 80%, and most preferably at least 90%of the liposomes are within the designated range.

In another aspect, the invention relates to methods for immunizing ananimal, preferably a mammal and most preferably a human, with one ormore immunogenic polypeptides or carbohydrates of interest. Thesemethods comprise:

delivering to said animal by a parenteral or enteral route an effectiveamount of a liposomal composition comprising:

a) an aqueous vehicle;

b) liposomes comprising

-   -   (i) dimyristoylphosphatidylcholine (“DMPC”),    -   (ii) dimyristoylphosphatidylglycerol (“DMPG”),        dimyristoyltrimethylammonium propane (“DMTAP”), or both DMPG and        DMTAP, and    -   (iii) at least one sterol derivative; and

c) one or more immunogenic polypeptide(s) or carbohydrate(s) covalentlylinked to between 1% and 100% of said at least one sterol derivative.

Preferred liposomal compositions, methods for making such compositions,steroid derivatives, methods for covalently coupling immunogenicpolypeptides or carbohydrates to such derivatives, and covalent linkagesare discussed in detail above and hereinafter.

Preferred enteral routes of administration include delivery by mouth(oral), nasal, rectal, and vaginal routes. Preferred parenteral routesof administration include intravenous, intramuscular, subcutaneous, andintraperitoneal routes.

In certain embodiments, the methods of the present invention comprisemultiple deliveries of an immunogenic polypeptide or carbohydrate,commonly referred to as “prime/boost” immunization protocol. Inpreferred embodiments, one or more of the prime and boost deliveriescomprises delivering to the animal by a parenteral or enteral route aliposomal composition of the present invention. In such immunizationprotocols, a priming delivery may be via a different route ofadministration than one or more boost deliveries. For example, a primingdelivery may be made by subcutaneous delivery of an immunogen, and aboost delivery may be made by intramuscular delivery.

In addition, the prime and one or more boost deliveries of an antigen ofinterest may be “homologous,” meaning that both the prime and boostcomprises delivery of a liposomal composition of the invention; or maybe “heterologous,” meaning that one of the prime or boost deliveriescomprises delivery of a liposomal composition of the present invention,while another delivery may be made by means of a different vaccineplatform. Such alternative vaccine platforms include, but are notlimited to, delivery of antigen in a non-liposomal vaccine formulation,delivery of DNA vaccine encoding the antigen, delivery of a recombinantviral vaccine, etc.

It is to be understood that the invention is not limited in itsapplication to the details of construction and to the arrangements ofthe components set forth in the following description or illustrated inthe drawings. The invention is capable of embodiments in addition tothose described and of being practiced and carried out in various ways.Also, it is to be understood that the phraseology and terminologyemployed herein, as well as the abstract, are for the purpose ofdescription and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts lipid structures used in exemplary embodiments. Rindicates the location of attachment of antigens to the lipids.

FIG. 2 depicts HIV-1 MPR epitopes N-MPR and C-MPR.

FIG. 3 depicts effects of the attached lipid moiety on MPR epitopesecondary structure analysis.

FIG. 4 depicts effects of the attached lipid moiety on MPR partitioninginto lipid bilayers.

FIG. 5 depicts induction of anti-peptide antibodies by N-MPR lipidconjugates.

FIG. 6 depicts induction of anti-peptide antibodies by N-MPRantigen-lipid conjugates.

FIG. 7 depicts induction of anti-peptide antibodies by C-MPRantigen-lipid conjugates.

FIG. 8A depicts the effect of attachment location of MPR epitopes toCHEMS on immunogenicity. FIG. 8B depicts the ability of lipid conjugatesof MPR to elicit antibodies that bind to recombinant gp140.

FIG. 9 depicts survival of animals immunized with M2eA1 protein ofinfluenza A (H1N1) virus on viral challenge.

DETAILED DESCRIPTION OF THE INVENTION

The goal of vaccine formulation is provide a combination of antigens andadjuvants capable of generating a sufficient population of memory Tcells and B cells to react quickly to a pathogen, tumor cell, etc.,bearing an antigen of interest. The present invention relates to methodsfor providing liposomal vaccine compositions, methods for themanufacture thereof, and methods for the use thereof to stimulate animmune response in an animal, which can meet this goal.

As discussed above, the liposomal formulations of the present inventioncomprise liposomes prepared using dimyristoylphosphatidylcholine(“DMPC”); together with dimyristoylphosphatidylglycerol (“DMPG”),dimyristoyltrimethylammonium propane (“DMTAP”), or both DMPG and DMTAP;and one or more sterol derivatives as a lipid anchor for an antigen ofinterest. The components of the liposomes may be naturally occurring orsynthetic.

Sterols are also known as steroid alcohols. They are a subgroup ofsteroids with a hydroxyl group at the 3-position of the A-ring. As notedabove, the term “sterol derivative” as used herein refers to anymolecule having the 4-member ring structure characteristic of steroidsand a hydroxyl (—OH) or ester (—OR) substitution at the 3-carbonposition. These may be purchased commercially, as in the case of certainsterol chloroformate derivatives, or may be prepared according tomethods well known in the art. See, e.g., WO/2000/075165;WO/2002/066490; U.S. Pat. Nos. 5,004,737; and 7,312,206.

The terms “antigenic polypeptide” and “antigenic carbohydrate” as usedherein refer to a polypeptide or carbohydrate, respectively, that isforeign to an animal and that, and upon delivery to an animal using, inwhole or part, the liposomal formulations described herein, stimulatesthe formation of antigen specific antibodies and/or an antigen-specificT-cell response. Antigenic polypeptides and/or carbohydrates, which maybe used in practicing the present invention, may be derived from, by wayof example only, viral pathogens, bacterial toxins, bacterial pathogens,fungal pathogens, cancer cells.

Methods for covalently linking an immunogenic polypeptide orcarbohydrate to a sterol derivative are well known in the art. Chemicalcross-linkers are discussed in numerous books and catalogues. See, e.g.,Wong, Chemistry of Protein Conjugation and Cross-linking, CRC Press,Boca Raton, Fla., 1991. These reagents often employ functional groupsthat couple to amino acid side chains of peptides. Designing across-linker involves selection of the functional moieties to beemployed. The choice of functional moieties is entirely dependent uponthe target sites available on the species to be crosslinked. Somespecies (e.g., proteins) may present a number of available sites fortargeting (e.g., lysine ε-amino groups, cysteine sulfhydryl groups,glutamic acid carboxyl groups, etc.), and selection of a particularfunctional moiety for inclusion in a sterol derivative may be madeempirically in order to best preserve a biological property of interest(e.g., binding affinity of an antibody, catalytic activity of an enzyme,etc.)

Coupling Through Amine Groups:

Imidoester and N-hydroxysuccinimidyl (“NHS”) esters are typicallyemployed as amine-specific functional moieties. NHS esters yield stableproducts upon reaction with primary or secondary amines. Coupling isefficient at physiological pH, and NHS-ester cross-linkers are morestable in solution than their imidate counterparts. HomobifunctionalNHS-ester conjugations are commonly used to cross-link amine-containingproteins in either one-step or two-step reactions. Primary amines arethe principle targets for NHS-esters. Accessible α-amine groups presenton the N-termini of proteins react with NHS-esters to form amides.However, because α-amines on a protein are not always available, thereaction with side chains of amino acids become important. While fiveamino acids have nitrogen in their side chains, only the ε-amino groupof lysine reacts significantly with NHS-esters. A covalent amide bond isformed when the NHS-ester cross-linking agent reacts with primaryamines, releasing N-hydroxysuccinimide.

Coupling Through Sulfhydryl Groups:

Maleimides, alkyl and aryl halides, α-haloacyls, and pyridyl disulfidesare typically employed as sulfhydryl-specific functional moieties. Themaleimide group is specific for sulfhydryl groups when the pH of thereaction mixture is kept between pH 6.5 and 7.5. At pH 7, the reactionof the maleimides with sulfhydryls is 1000-fold faster than with amines.Maleimides do not react with tyrosines, histidines or methionines. Whenfree sulfhydryls are not present in sufficient quantities, they canoften be generated by reduction of available disulfide bonds.

Coupling Through Carboxyl Groups:

Carbodiimides couple carboxyls to primary amines or hydrazides,resulting in formation of amide or hydrazone bonds. Carbodiimides areunlike other conjugation reactions in that no cross-bridge is formedbetween the carbodiimide and the molecules being coupled; rather, apeptide bond is formed between an available carboxyl group and anavailable amine group. Carboxy termini of proteins can be targeted, aswell as glutamic and aspartic acid side chains. In the presence ofexcess cross-linker, polymerization may occur because proteins containboth carboxyls and amines. No cross-bridge is formed, and the amide bondis the same as a peptide bond, so reversal of the cross-linking isimpossible without destruction of the protein.

Nonselective Reactive Groups:

A photoaffinity reagent is a compound that is chemically inert butbecomes reactive when exposed to ultraviolet or visible light.Arylazides are photoaffinity reagents that are photolyzed at wavelengthsbetween 250-460 nm, forming a reactive aryl nitrene. The aryl nitrenereacts nonselectively to form a covalent bond. Reducing agents must beused with caution because they can reduce the azido group.

Coupling Through Arginines:

Glyoxals are useful compounds for targeting the guanidinyl portion ofarginine residues. Glyoxals will target arginines at mildly alkaline pH.There is some cross-reactivity (the greatest at higher pH) with lysines.

Coupling Through Carbonyl Groups:

Carbonyls (aldehydes and ketones) react with amines and hydrazides at pH5-7. The reaction with hydrazides is faster than with amines, makingthis useful for site-specific cross-linking. Carbonyls do not readilyexist in proteins; however, mild oxidation of sugar moieties usingsodium metaperiodate will convert vicinal hydroxyls to aldehydes orketones. For carbohydrates with reducing end(s), the carbonyl group(s)can be reactive towards a hydrazine moiety to form a hydrazone bond.S-HyNic is a heterobifunctional linker used to incorporate HyNic(6-hydrazinonicotinamide) moieties into molecules through a free aminogroup via an activated ester (i.e. NHS). The addition of a HyNichydrazine linker permits formation of a conjugate in slightly acidicbuffer (100 mM NaPO4, pH6). For carbohydrates without a reducing end,CDAP specific activation may be used. Under mild conditions (pH 9.5 foractivation and pH 7 for conjugation), 1-cyano-4-dimethylaminopyridiniumtetrafluoroborate (“CDAP”) converts hydroxyl groups to cyanyl esterswhich will then form carbamates in the presence of amine groups.

A functional moiety can be attached directly to a ring atom on thepolycyclic sterol nucleus, or may be attached to the sterol nucleusthrough one or more linking atoms. An exemplary covalent linkage betweena sterol and a reactive group has the structure St-R3-X, wherein:

-   St is a ring atom of the sterol derivative;-   R3 is C₀₋₁₈ straight or branched chain alkyl, or C₀₋₁₂ straight or    branched chain alkyl-(alkylene oxide)_(n)-C₀₋₁₂ straight or branched    chain alkyl, wherein n is on average between 40 and 1000, wherein    each said straight or branched chain alkyl comprises from 0-4 chain    heteroatoms and one or more substituents independently selected from    the group consisting of halogen, trihalomethyl, —C₁₋₆ alkoxy, —NO₂,    —NH₂, —OH, —CH₂OH, —CONH₂, and —C(O)(OR4) where R4 is H or C₁₋₃    alkyl; and-   X is a reactive linking group, most preferably an amine linking    group, a sulfhydryl linking group, a carboxyl linking group, a    photoaffinity linking group, an arginine linking group, and a    carbonyl linking group.

The polymeric substances optionally included in the linkage chemistryare preferably poly(alkylene oxides). As used herein, the term “alkyleneoxide” refers to the structure, —X—O—, where X is an alkylene moietycovalently linked to oxygen O; thus poly(alkylene oxide) refers to thestructure —(X—O—)_(m))—. It is preferred that the poly(alkylene oxide)polymer be a nonbranched homopolymer (i.e., a polymer of the structure—((CH₂)_(n)—O—)_(m))—in which n does not vary) such as poly(ethyleneoxide) derived from ethylene glycol. Alternative polymers such as otherpolyalkylene oxide homopolymers (e.g., methylene oxide, propylene oxide,isopropylene oxide, and butylene oxide polymers) and co-polymers orblock co-polymers of poly(alkylene oxides) may also be used. In thoseaspects of the invention where PEG-based polymers are used, it ispreferred that they have average length n of between 40 and 1000monomeric units. Molar equivalent amounts of the other alkylene oxidesmay be determined readily by those of ordinary skill in the art toarrive at preferred average molecular weights for other homopolymers andcopolymers.

Average molecular weights of the present invention are measured usingthe “number-average” method. In a mixture of polymer molecules withdifferent molecular weights in which the number of molecules having aparticular molecular weight, M_(i), is given by N_(i) the“number-average” probability of a given mass being present is

$P_{i} = \frac{N_{i}}{\overset{\infty}{\sum\limits_{j = 0}}N_{j}}$and the number-average molecular weight is given by the formula

$\overset{\_}{M_{n}} = {{\sum\limits_{i = 0}^{\infty}{\left( \frac{N_{i}}{\sum\limits_{j = 0}^{\infty}N_{j}} \right)M_{i}}} = \frac{\sum\limits_{i = 0}^{\infty}{N_{i}M_{i}}}{\sum\limits_{j = 0}^{\infty}N_{j}}}$The number average is the simple arithmetic mean, representing the totalweight of the molecules present divided by the total number ofmolecules. The number-average molecular weight of a polymer may bemeasured by vapor pressure osmometry using methods and apparatuses wellknown to those of skill in the art.

Alternative polymeric substances which may be used in place ofpoly(alkylene oxides) include materials such as dextran, polyvinylpyrrolidones, polysaccharides, starches, polyvinyl alcohols, polyacrylamides or other similar polymers. Those of ordinary skill in the artwill realize that the foregoing is merely illustrative and not intendedto restrict the type of non-antigenic polymeric substances suitable foruse herein.

“Administration” as used herein with respect to an animal, includingpreferably a mammal and most preferably a human, refers to delivery ofan exogenous reagent to a cell, tissue, organ, or biological fluid ofthe subject.

“Effective amount” as used herein refers to an amount of a reagent thatcan ameliorate, reverse, mitigate, or prevent a symptom or sign of amedical condition or disorder. Unless dictated otherwise, explicitly orotherwise, an “effective amount” is not limited to a minimal amountsufficient to ameliorate a condition, or to an amount that results in anoptimal or a maximal amelioration of the condition. “Effective amount”within the context of administration of a vaccine is that which causesan immune response in the mammal. Such an effective amount may not be,in and of itself, sufficient to cause such an immune response, but maybe used together with previous or subsequent delivery of additionalreagents (e.g. a prime-boost vaccination). An “immunological response”or “immune response” as used herein encompasses at least one or more ofthe following effects: the production of antibodies by B-cells; and/orthe activation of suppressor T-cells and/or T-cells directedspecifically to an antigen or antigens present in the vectors,composition or vaccine of interest.

A variety of in vitro and in vivo assays are known in the art formeasuring an immune response, including measuring humoral and cellularimmune responses, which include but are not limited to standardimmunoassays, such as RIA, ELISA assays; intracellular staining; T cellassays including for example, lymphoproliferation (lymphocyteactivation) assays, CTL cytotoxic cell assays, or by assaying forT-lymphocytes specific for the antigen in a sensitized subject. Suchassays are well known in the art.

The preparation of liposomes is well known in the prior art. In general,liposomes have been made by a number of different techniques includingethanol injection (Batzri et al., Biochem. Biophys. Acta. 298:1015,1973); ether infusion (Deamer et al., Biochem. Biophys. Acta. 443:629,1976; Schieren et al., Biochem. Biophys. Acta. 542:137, 1978); detergentremoval (Razin, Biochem. Biophys. Acta. 265:24 1972); solventevaporation (Matsumato et al., J. Colloid Interface Sci. 62:149, 1977);evaporation of organic solvents from chloroform in water emulsions(REV's) (Szoka Jr. et al., Proc. Natl. Acad. Sci. USA, 75:4194, 1978);extrusions of MLVs or EUV's through a nucleopore polycarbonate membrane(Olson et al., Biochem. Biophys. Acta. 557:9, 1979); freezing andthawing of phosopholipid mixtures (Pick, Arch. Biochem. Biophys.,212:186, 1981), as well as sonication and homogenization. By convention,liposomes are categorized by size, and a 3-letter acronym is used todesignate the type of liposome being discussed. Multilamellar vesiclesare generally designated “MLV.” Small unilamellar vesicles aredesignated “SUV,” and large unilamellar vesicles are designated “LUV.”These designations are sometimes followed by the chemical composition ofthe liposome. For a discussion of nomenclature and a summary of knowntypes of liposomes, see Storm et al., PSIT, 1: 19-3, 1998.

The liposomal compositions of the invention may further comprise, eitheras part of the liposome itself or as part of the vehicle in which theliposomes are suspended, various excipients, adjuvants, carriers,auxiliary substances, modulating agents, and the like.

A carrier, which is optionally present, is a molecule that does notitself induce the production of antibodies harmful to the individualreceiving the composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycollic acids, polymeric amino acids, amino acidcopolymers, lipid aggregates (such as oil droplets or liposomes), andinactive virus particles. Examples of particulate carriers include thosederived from polymethyl methacrylate polymers, as well as microparticlesderived from poly(lactides) and poly(lactide-co-glycolides), known asPLG. See, e.g., Jeffery et al., Pharm. Res. 10:362, 1993; McGee et al.,J. Microencapsul. 14: 197, 1997; O'Hagan et al., Vaccine 11:149, 1993.Such carriers are well known to those of ordinary skill in the art.

Adjuvants include, but are not limited to: (1) aluminum salts (alum),such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.;(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (see below) orbacterial cell wall components), such as for example (a) MF59(International Publication No. WO 90/14837), containing 5% Squalene,0.5% Tween 80, and 0.5% Span 85 (optionally containing various amountsof MTP-PE (see below), although not required) formulated into submicronparticles using a microfluidizer such as Model 11OY microfluidizer(Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4%Tween 80, 5% pluronic-blocked polymer L1 21, and MDP eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.); (3) one or more bacterial cell wallcomponents from the group consisting of monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (Detoxu); (4) saponin adjuvants, such as Stimulon™ (CambridgeBioscience, Worcester, Mass.); (5) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (6) cytokines, such as interleukins(IL-I, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), beta chemokines (MIP, 1-alpha, 1-beta Rantes,etc.); (7) detoxified mutants of a bacterial ADP-ribosylating toxin suchas a cholera toxin (CT), a pertussis toxin (PT), or an E. coliheat-labile toxin (LT), particularly LT-K63 (where lysine is substitutedfor the wild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S 109(where serine is substituted for the wild-type amino acid at position109), and PT-K9/G129 (where lysine is substituted for the wild-typeamino acid at position 9 and glycine substituted at position 129) (see,e.g., International Publication Nos. WO93/13202 and WO92/19265); and (8)other substances that act as immunostimulating agents to enhance theeffectiveness of the composition.

Preferred adjuvants include pathogen-associated molecular patterns(PAMPs), which mediate innate immune activation via Toll-like Receptors(TLRs), (NOD)-like receptors (NLRs), Retinoic acid inducible gene-based(RIG)-I-like receptors (RLRs), and/or C-type lectin receptors (CLRs).Examples of PAMPs include lipoproteins, lipopolypeptides,peptidoglycans, zymosan, lipopolysaccharide, neisserial porins,flagellin, profillin, α-galactosylceramide, muramyl dipeptide.Peptidoglycans, lipoproteins, and lipoteichoic acids are cell wallcomponents of Gram-positive. Lipopolysaccharides are expressed by mostbacteria, with MPL being one example. Flagellin refers to the structuralcomponent of bacterial flagella that is secreted by pathogenic andcommensal bacterial. α-Galactosylceramide (α-GalCer) is an activator ofnatural killer T (NKT) cells. Muramyl dipeptide is a bioactivepeptidoglycan motif common to all bacteria

Other preferred adjuvants include viral double-stranded RNA, which issensed by the intracellular receptor TLR3; CpG motifs present onbacterial or viral DNA or ssRNA, which are sensed by TLR7, 8, and 9;all-trans retinoic acid; and heat shock proteins such as HSP70 and Gp96,which are highly effective carrier molecules for cross-presentation.Pharmaceutical adjuvants include resiquimod, a TLR7/8 agonists, andimiquimod, a TLR7 agonist.

The liposomes of the present invention are preferably formulated aspharmaceutical compositions for parenteral or enteral delivery. Atypical pharmaceutical composition for administration to an animalcomprises a pharmaceutically acceptable vehicle such as aqueoussolutions, non-toxic excipients, including salts, preservatives, buffersand the like. See, e.g., Remington's Pharmaceutical Sciences, 15th Ed.,Easton ed., Mack Publishing Co., pp 1405-1412 and 1461-1487 (1975); TheNational Formulary XIV, 14th Ed., American Pharmaceutical Association,Washington, D.C. (1975). Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, vegetable oil and injectable organic esterssuch as ethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to routine skills in the art.

Repeated administrations of a particular vaccine (homologous boosting)have proven effective for boosting humoral responses. Such an approachmay not be effective at boosting cellular immunity because priorimmunity to the vector tends to impair robust antigen presentation andthe generation of appropriate inflammatory signals. One approach tocircumvent this problem has been the sequential administration ofvaccines that use different antigen-delivery systems (heterologousboosting).

In a heterologous boosting regimen, at least one prime or boost deliverycomprises delivery of the liposomal formulations described herein. Theheterologous arm of the regimen may comprise delivery of antigen usingone or more of the following strategies:

-   attenuated and/or inactivated bacteria or viruses comprising the    antigen of interest, which are particles that have been treated with    some denaturing condition to render them ineffective or inefficient    in mounting a pathogenic invasion;-   purified antigens, which are typically naturally-produced antigens    purified from a cell culture of the pathogen or a tissue sample    containing the pathogen, or a recombinant version thereof;-   live viral or bacterial delivery vectors recombinantly engineered to    express and/or secrete antigens in the host cells of the subject.    These strategies rely on genetically engineering the viral vectors    to be non-pathogenic and non-toxic;-   antigen presenting cell (APC) vectors, such as a dendritic cell (DC)    vector, which comprise cells that are loaded with an antigen, or    transfected with a composition comprising a nucleic acid encoding    the antigen;-   tumor cells, for example, autologous and allogeneic tumor cells; and-   naked DNA vectors and naked RNA vectors which may be administered by    a gene gun, electroporation, bacterial ghosts, microspheres,    microparticles, liposomes, polycationic nanoparticles, and the like.

A prime vaccine and a boost vaccine can be administered by any one orcombination of the following routes. In one aspect, the prime vaccineand boost vaccine are administered by the same route. In another aspect,the prime vaccine and boost vaccine are administered by differentroutes. The term “different routes” encompasses, but is not limited to,different sites on the body, for example, a site that is oral, non-oral,enteral, parenteral, rectal, intranode (lymph node), intravenous,arterial, subcutaneous, intramuscular, intratumor, peritumor,intratumor, infusion, mucosal, nasal, in the cerebrospinal space orcerebrospinal fluid, and so on, as well as by different modes, forexample, oral, intravenous, and intramuscular.

An effective amount of a prime or boost vaccine may be given in onedose, but is not restricted to one dose. Thus, the administration can betwo, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more, administrations of the vaccine. Where there is morethan one administration of a vaccine the administrations can be spacedby time intervals of one minute, two minutes, three, four, five, six,seven, eight, nine, ten, or more minutes, by intervals of about onehour, two hours, three, four, five, six, seven, eight, nine, ten, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and so on. Inthe context of hours, the term “about” means plus or minus any timeinterval within 30 minutes. The administrations can also be spaced bytime intervals of one day, two days, three days, four days, five days,six days, seven days, eight days, nine days, ten days, 11 days, 12 days,13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,21 days, and combinations thereof. The invention is not limited todosing intervals that are spaced equally in time, but encompass doses atnon-equal intervals, such as a priming schedule consisting ofadministration at 1 day, 4 days, 7 days, and 25 days, just to provide anon-limiting example.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The examples providedherein are representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention.

EXAMPLE 1 Liposomes Conjugated to HIV-1 Gp41

Despite extensive research, attempts to elicit broadly neutralizingantibodies (bnAb) to HIV have not yet succeeded. The membrane proximalregion (MPR) of HIV-1 gp41 is a desirable target for development of avaccine that elicits neutralizing antibodies since the patient-derivedmonoclonal antibodies, 2F5 and 4E10, bind to MPR and neutralize primaryHIV isolates. It has been demonstrated that two antibodies, designated2F5 and 4E10, cross-react with lipids, and structural studies suggestthat MPR immunogens may be presented in a membrane environment. However,efforts to date have not succeeded in eliciting antibodies with thebreadth or potency of patient-derived antibodies.

The lipid reactivities of bnAb 2F5 and 4E10 have been a topic of intensestudy. Both antibodies have unusually long, hydrophobic CDRH3 regionsand cross-react with phospholipids and other autoantigens. Moreover,biophysical models suggest that the MPR intercalates into the membranein native virions. These observations have led to suggestions that MPRimmunogens may be presented optimally in a lipid bilayer environment.The majority of strategies to insert the epitopes in a lipid environmenthave involved chimeric viruses or liposomal formulations of recombinantconstructs with transmembrane peptide domains. Additionally, variationsin lipid membrane composition appear to alter MPR peptide accessibility,and modulation of the peptide anchoring mechanism may exert similareffects.

We hypothesized that covalent attachment of lipid anchors would enhancethe humoral immune response to MPR-derived peptides presented inliposomal bilayers. Three peptides were selected, corresponding to the2F5 epitope (N-MPR), the 4E10 epitope (C-MPR) and a helicallyconstrained peptide spanning both epitopes (NC-MPR). These epitopes aresummarized in FIG. 1. We systematically examined the effects of thelipid anchors on the humoral response in mice immunized with thelipopeptides in liposomes.

A. Materials and Methods

Amino acid building blocks, resins and coupling agents were obtainedfrom Novabiochem (Darmstadt, Germany), Anaspec (San Jose, Calif.) orChemPep (Miami, Fla.). Cholesterol, dimyristoylphosphatidylcholine(DMPC), dimyristoylphosphatidylglycerol (DMPG), oxidizedphosphatidylcholine (PC; #870601), brain sphingomyelin (SM; #860082) andtetramyristoylcardiolipin (CL; #710332) were obtained from Avanti PolarLipids (Alabaster, Ala.). Dipalmitoylphosphatidylethanolamine (PE;#LP-R4-019) and dipalmitoylglycerol (DPG; #LP-R4-028) were obtained fromGenzyme Pharmaceuticals (Cambridge, Mass.). Palmitic acid (PA; #P5585)and 5-cholenic acid-3β-ol (CHOL; #C2650) were obtained fromSigma-Aldrich (St. Louis, Mo.). Anhydrous solvents of 99.8% or greaterpurity were obtained from Acros Organics (Geel, Belgium). Monophosphoryllipid A derived from Escherichia coli (MPL; #L6638) was obtained fromSigma-Aldrich. 2F5 and 4E10 monoclonal antibodies were obtained throughthe NIH AIDS Research and Reference Reagent Program, Division of AIDS,NIAID, NIH from Dr. Hermann Katinger. Unless otherwise specified, allother reagents were obtained from Sigma-Aldrich.

i. Lipopeptide Synthesis

Peptides were synthesized on NovaPEG resin in an automated solid phasesynthesizer (ABI 433A, Applied Biosystems, Foster City, Calif.) withstandardfluorenylmethyloxycarbonyl/o-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate/n-hydroxybenzotriazole(FMOC/HBTU/HOBT) protocols. When appropriate, an orthogonally protectedlysine(Fmoc-Lys(1-(4,4-Dimethyl-2,6-dioxo-cyclohexylidene)-3-methyl-butyl)-OH;Fmoc-Lys(ivDde)-OH) was incorporated at the C terminus for on-resinconjugation of lipids or biotin. The N terminus was generallyBoc-protected unless the peptide was intended for N terminalmodification, in which case Fmoc protection was utilized. Removal of theivDde group was accomplished by 3×15 minute treatments of the peptidylresin with 2% hydrazine hydrate in dimethylformamide (DMF; 10 mL per gresin). The resin was washed in DMF (3×10 mL) and dichloromethane (DCM;3×10 mL) and dried under vacuum.

Nomenclature and structures of lipids used in this study are summarizedin FIG. 1. Lipid conjugation was accomplished via amidation of acarboxylated lipid and a deprotected lysine ε-amine at the C terminus.For N terminal conjugation, lipids were attached directly to thedeprotected N terminus. Several of the lipids contained carboxyl groups.In the case of DPG, PE, SM, and CL, a carboxyl group was introduced viareaction of an available alcohol (DPG, SM, CL) or amine (PE) withsuccinic anhydride. For DPG-Suc, 1.8 mmol DPG was dissolved in 5 mLanhydrous DCM and combined with 3.6 mmol succinic anhydride in 10 mLanhydrous pyridine. The mixture was refluxed at 60° C. overnight. ForPE-Suc, 1.5 mmol PE was combined with 3 mmol succinic anhydride and 6mmol triethylamine in 50 mL anhydrous chloroform (CHCl₃). The mixturewas stirred at room temperature overnight. For CL-Suc, 80 μmol CL wascombined with 400 μmol succinic anhydride and 400 μmol triethylamine in5 mL anhydrous CHCl₃. The mixture was refluxed at 60° C. overnight. ForSM-Suc, 136 μmol SM was combined with 684 μmol succinic anhydride and684 μmol triethylamine in 5 mL anhydrous CHCl₃. The mixture was refluxedat 60° C. overnight. Reactions were continued to completion as monitoredby thin layer chromatography (TLC) and matrix-assisted laserdesorption/ionization mass spectrometry (MALDI-MS; Voyager D E, AppliedBiosystems, Foster City, Calif.) in para-nitroaniline matrix. Productswere washed twice with 1M hydrochloric acid (HCl), dried over sodiumsulfate and stored dry until use. Carboxylated lipids were obtained inapproximately 90-100% yield. Molecular weights and TLC R_(F) values wereas follows: DPG-Suc, 668.19 Da, R_(F) 0.71 in 20:1 DCM:acetone; PE-Suc,790.02 Da, R_(F) 0.81 in 65:25:4 CHCl₃:MeOH:NH₄OH; CL-Suc, 1335.90 Da,R_(F) 0.24 in 65:25:4 CHCl₃:MeOH:NH₄OH; SM-Suc, 767.83 Da, 826.62 Da,853.02 Da, 910.78 Da, R_(F) 0.66-0.79 in 65:25:4 CHCl₃:MeOH:NH₄OH.SM-Suc gave a series of peaks because the starting material was anatural product with a distribution of aliphatic chain lengths.

Lipidation was accomplished by activation of 270 μmol carboxylated lipidwith 270 μmol each of HBTU, HOBT and diisopropylethylamine (DIEA) inanhydrous DMF/DCM (DCM as needed for lipid solubilization) for 30 min atroom temperature followed by addition of 67.5 μmol resin and continuedreaction under argon for 24 h at room temperature. Following thereaction, the resin was washed with DMF (4×10 mL) and DCM (4×10 mL) toremove unreacted lipids and dried under vacuum. Peptides were cleavedfrom the resin by treatment with trifluoroacetic acid containing 2.5%water, 2.5% ethanedithiol and 1% triisopropylsilane for 4 hours underargon. Cleaved peptides were precipitated into cold ethyl ether. Theprecipitate was pelleted by centrifugation at 3000 rpm (RT6000, Sorvall,Waltham, Mass.) and washed once with cold ethyl ether. The ether waspoured off and the pellet was re-dissolved in methanol (MeOH),transferred to a round bottom flask, dried by rotary evaporation underreduced pressure and further dried under high vacuum. Lipopeptides werefurther separated from unconjugated peptide by reverse phase highpressure liquid chromatography (RP-HPLC; DX 500, Dionex, Sunnyvale,Calif.) on a semi-preparative C4 column (214TP510, Grace Vydac,Deerfield, Ill.) until unconjugated peptide was no longer detectable byMALDI-MS. Lipopeptide fractions were identified by MALDI-MS in2,5-dihydroxybenzoic acid matrix, pooled and lyophilized. Stocklipopeptide solutions were prepared in MeOH or MeOH/CHCl₃ and stored at−20° C. Final yields were approximately 5-10%.

Biotinylated peptides were prepared for use in ELISA by an analogousmethod. Biotin was attached to the deprotected C terminal amine byactivation of 500 μmol D-biotin with 500 μmol HBTU/HOBT/DIEA in 1.65 mLanhydrous 1:1 DMF/dimethylsulfoxide (DMSO) for 30 min followed byaddition of resin and continued reaction under argon for 24 h at roomtemperature. Following the reaction, the resin was washed with 1:1DMF/DMSO (3×10 mL), DMF (3×10 mL) and DCM (3×10 mL) and dried undervacuum. Biotinylated peptides were cleaved and purified as describedabove. Biotin content was quantified by4′-hydroxyazobenzene-2-carboxylic acid dye exclusion (Sigma #H2153)according to the manufacturer's instructions.

ii. Liposome Preparation

Lipopeptides were formulated in liposomes composed of 15:2:3:0.3DMPC:DMPG:Cholesterol:MPL. Prior to use, glassware was rinsed with MeOHand CHCl₃ and dried for at least 90 mM at 150° C. to destroy pyrogens.Lipid solutions were combined in borosilicate glass tubes and dried to athin film by rotary evaporation under reduced pressure. Films werefurther dried under high vacuum overnight. Lipids were hydrated insterile PBS (UCSF Cell Culture Facility) by intermittent vortexing andbath sonication under argon for a brief period (approximately 15seconds) to disperse the lipids into the buffer. Defined diametervesicles were formed by extrusion 11 times through 400 nm polycarbonatemembranes using a hand-held extruder (Avestin, Ottowa, Canada). Toprevent contamination, the extruder was disassembled and thoroughlycleaned with MeOH and sterile PBS between samples. The final formulationcontained 1 mg/mL lipopeptide and 0.5 mg/mL monophosphoryl lipid A in 20mM carrier lipid. Vesicle size was characterized by dynamic lightscattering (Zetasizer 3000, Malvern, New Bedford, Mass.). Liposomes werestored at 4° C. under argon until use.

iii. Circular Dichroism

Liposomal lipopeptide samples were prepared as described above with thefollowing modifications. Stock liposome solutions containing 5 mMcarrier lipid and 500 μM lipopeptide were prepared in 10 mM phosphate,pH 7.4. To minimize light scattering, liposomes were prepared by bathsonication under argon until a size of less than 100 nm was obtained.For analysis, samples were diluted to 5 μM lipopeptide in 10 mMphosphate buffer containing 1 mM carrier lipid. Spectra were obtainedwith a J-715 spectrapolarimeter (Jasco, Easton, Md.) and data wereprocessed using Jasco software. Data were acquired in continuousscanning mode with a pathlength of 1 cm, 0.1 nm interval and scan speedof 1 nm/s. Each spectrum represents an average of two scans. Abackground spectrum of “empty” liposomes in buffer was subtracted fromeach sample spectrum. Percent helicity was estimated from θ₂₂₂ accordingto the method of Taylor and Kaiser.

iv. Tryptophan Fluorescence

Lipopeptide membrane partitioning was characterized by measurement oftryptophan fluorescence intensity as described with modifications.Briefly, DMPC:DMPG:Cholesterol liposomes were prepared inphosphate-buffered saline as described above. Lipopeptide stocksolutions were prepared in MeOH. 12 nmol lipopeptide was injected viaglass syringe (Hamilton, Reno, Nev.) into 1.2 mL buffer containingdiluted liposomes (10-150 μM lipid). The samples were mixed by inversionand allowed to equilibrate in the dark at room temperature overnight.Fluorescence emission spectra were obtained on a SPEX Fluorologspectrophotometer (Horiba Jobin Yvon, Edison, N.J.) with 1 cmpathlength, 2.5 mm excitation slit, 5.0 mm emission slit, 1 sintegration time and 1 nm interval. For each liposome concentration, abackground spectrum of “empty” liposomes in buffer was subtracted fromthe sample spectrum. Fluorescence intensity was determined byintegration of the tryptophan fluorescence peak and data were normalizedto the highest intensity in each sample series. Partition coefficientswere calculated from the double reciprocal plot of normalizedfluorescence intensity versus lipid concentration, according to theequation F=(F₀*L*K_(p))/(55.6+K_(p)*L) [29].

v. Animal Immunizations

All animal procedures were conducted in accordance with the policies andapproval of the appropriate Institutional Animal Care and Use Committee.8 week-old female BALB/C mice (Jackson Laboratories, Bar Harbor, Me.)were housed in a pathogen-free barrier facility. Animals receivedsubcutaneous immunizations in alternating hind hocks on Days 0 and 14.Each injection contained 50 μg lipopeptide, 25 μg MPL and 1 μmol lipidvehicle in 50 μL sterile phosphate-buffered saline. On Day 28 blood wascollected from the submandibular vein for characterization of antibodyresponses. Cells were removed by centrifugation at 14,000 rpm for 15 min(5415C, Eppendorf, Westbury, N.Y.) and sera were stored at −80° C. untiluse.

vi. ELISA

ELISAs were developed to quantify binding of immune sera to peptides,lipids, and recombinant gp140. Peptide ELISAs were conducted using MPRpeptides biotinylated as described above and captured on 96 wellstreptavidin-coated plates (#15120, Pierce, Rockford, Ill.). Assays wereperformed according to the manufacturer's instructions withmodifications. Biotinylated peptides were added to wells in PBScontaining 0.1% Tween-20 (PBS-T) and incubated for 2 hr at 37° C.Following a wash step, sera were serially diluted in PBS containing 0.1%casein (C7078, Sigma-Aldrich) (PBS-C), added to wells and incubated for30 min at 37° C. After reconstitution, horseradish peroxidase-conjugatedsecondary antibodies (IgG, IgG1, IgG2a; Jackson Immunoresearch, WestGrove, Pa.) were diluted 1:1 in glycerol for long-term storage at −20°C. and further diluted 1:1000 in PBS-C immediately prior to use.Following a wash step, secondary antibodies were added to wells andincubated for 30 min at 37° C. Following a final wash step, atetramethylbenzidine substrate solution (#T0440, Sigma-Aldrich) wasadded to wells and incubated for 30 min at room temperature. Thereaction was stopped with 0.5M H₂SO₄ and the yellow product wasmonitored at 450 nm (Optimax, Molecular Devices, Sunnyvale, Calif.). Allincubations were done in 100 μL volumes and wells were washed 6 timeswith PBS-T between each step. Titer was defined as the reciprocaldilution of immune sera yielding an optical density twice that of 1:200preimmune sera after subtraction of background wells lacking serum.IgG1/IgG2a ratios were calculated as an average of optical densityquotients measured at 3 dilutions after subtraction of backgroundvalues. All samples were assayed in duplicate.

Lipid ELISAs were performed generally as follows. Lipids were diluted to0.2 mg/mL in EtOH and 50 μL per well were added to flat-bottomeduntreated polystyrene plates (Fisher) and allowed to dry overnight.Plates were blocked with 0.5% casein for 2 hr. After a wash step, immunesera were diluted 1:200 in 10% fetal bovine serum in PBS and incubatedin wells for 1 hr. Wells were washed and peroxidase-conjugatedanti-mouse IgG was diluted 1:1000 in PBS-C and added to wells for 1 hr.Following a wash step, plates were then read as indicated above. Allincubations were done in 100 μL volumes at room temperature and wellswere washed 6 times with PBS between each step.

Recombinant gp140 ELISAs were performed follows: Ba-1 gp140 (ImmuneTechnology Corp, New York, N.Y.) was diluted to 5 μg/mL in 50 mM sodiumcarbonate, pH 9.6 and 100 μL per well were added to flat-bottomed highcapacity immunoassay plates (Costar). Plates were sealed with parafilmand incubated at 4° C. overnight. Plates were blocked with 0.5% caseinfor 2 hr. After a wash step, immune sera were diluted 1:50 in PBS-C andincubated in wells for 1 hr. Wells were washed and peroxidase-conjugatedanti-mouse IgG was diluted 1:1000 in PBS-C and added to wells for 30min. Following a wash step, plates were then developed and read asindicated above. All incubations were done in 100 μL volumes at 37° C.and wells were washed 6 times with PBS-T between each step.

vii. Statistical Analysis

Statistical significance was assessed by analysis of variance andtwo-tailed Student's t test. Differences were considered significant ifthey exhibited p values <0.05 in the Student's t test. Data analyseswere performed using Microsoft Excel and SigmaPlot.

B. Results

i. Preparation and Analysis of Lipopeptides and Liposomes

This study sought to address the role of lipid structure in the humoralimmune response to MPR lipopeptides formulated in liposomes. Threepeptides were selected for lipid modification, corresponding to the 2F5epitope (N-MPR), the 4E10 epitope (C-MPR) and an extended peptidespanning both epitopes (NC-MPR; summarized in FIG. 1). The sequences ofN-MPR and C-MPR included flanking residues that were found to maximizebinding affinities for their respective antibodies in vitro. Twohelix-promoting isobutyric acid residues were incorporated into NC-MPR,as previously implemented in the design of a helically constrained 4E10epitope peptide. The N terminus of NC-MPR was extended to include thefull 2F5 epitope. An orthogonally protected lysine was included forlipid conjugation at the C terminus to mimic the native structure, inwhich the C terminus is anchored to the membrane.

Lipid anchors were selected to represent several basic lipid types:fatty acids, diacylglycerols, phospholipids and sterols. Additionally,some are implicated in cross-reactivity with 4E10 and 2F5 (cardiolipin)or in virus-cell fusion (virion lipid phosphatidylethanolamine; raftlipids sphingomyelin and cholesterol). Consideration was also given tolipid anchors that may facilitate elicitation of antibodies binding toboth peptide and lipid moieties. Specifically, lipids lacking aphosphate (palmitic acid and diacylglycerol) were selected forcomparison to phosphate-containing lipids because the phosphate and headgroup moieties are important in recognition by anti-phospholipidantibodies. Cholenic acid (CHOL) was chosen in addition to cholesterolhemisuccinate (CHEMS) due to work indicating that the 3β-hydroxyl is aprimary moiety responsible for recognition of cholesterol byanti-cholesterol antibodies.

For those lipids lacking a carboxyl group, one was introduced byreaction with succinic anhydride. For peptide conjugation, the on-resinlipidation strategy allowed complete removal of unreacted lipid viaextensive washing of the resin prior to cleavage. The remainingcontaminant, unreacted peptide, was removed by RP-HPLC. Conjugatedpeptides were obtained in approximately 5-10% yield; steric hinderancein modification of the C terminal lysyl ε-amine and loss upon RP-HPLCpurification may have contributed to the relatively poor yield. Humanmonoclonal antibodies 2F5 and 4E10 bound strongly to biotinylated MPRpeptides containing their epitopes (N-MPR and C-MPR, respectively) byELISA (FIG. 2). The cause for weak binding of 2F5 to C-MPR is uncertainbut may be attributed to partial overlap in the peptide sequences.Regardless, sera of mice immunized with N-MPR lipopeptides did not bindto C-MPR by ELISA and vice versa. Liposomal formulation of MPRlipopeptides resulted in vesicles approximately 175-250 nm in diameter.Addition of peptide or lipopeptide did not appreciably affect vesiclesize, with the exception of N-MPR-PE liposomes, which were slightlysmaller than the others.

When formulated in liposomes, N-MPR secondary structure was greatlyaltered by the attached lipid moiety (FIG. 3a ). Whereas attachment ofCHEMS to N-MPR resulted in a modest increase in helicity (26.5% versus20.7%), attachment of DPG substantially increased helical content (47.8%versus 20.7%). In contrast, attachment of CHEMS to NC-MPR only modestlyaffected its already helical conformation (FIG. 3b ). For NC-MPR, thedata suggest a trend in which C terminal attachment promotes helicity(8% and 5% respective increases when comparing ‘C Terminus’ versus‘Unconjugated’ and ‘Both Termini’ versus ‘N Terminus’), whereas Nterminal attachment decreases helicity (2% and 5% respective decreaseswhen comparing ‘N Terminus versus ‘Unconjugated’ and ‘Both Termini’versus ‘C Terminus’). The NC-MPR spectra are in agreement with thosereported for 4E10 epitope peptides with nearly identical C terminalhelix restraints. By comparison, the lower overall helicity of NC-MPRmay be attributed to the contribution of the extended N terminal segmentnot present in the peptide synthesized by Cardoso and coworkers.

Tryptophan fluorescence experiments revealed that the attached lipidmoiety also affects partitioning of N-MPR into lipid bilayers (FIG. 4).Both PA and CHEMS conjugates exhibited incremental differences intryptophan fluorescence as a function of liposome concentration. Thisindicates that as the concentration of liposomes is increased,additional lipopeptides partition into the membrane. However, tryptophanfluorescence of N-MPR-DPG was unaffected by increasing lipidconcentration over the range measured. The K_(p) of N-MPR-DPG wasestimated to be at least an order of magnitude greater than that ofN-MPR-PA or N-MPR-CHEMS (5.84×10⁸ versus 2.01×10⁷ and 1.95×10⁷,respectively). This observation suggests that N-MPR-DPG partitions morestrongly into bilayer membranes than the other conjugates.Alternatively, the possibility that DPG promotes self-aggregation cannotbe excluded. As hydrophobic bilayer environments are known to promotehelicity of peptides, the increased helicity of N-MPR-DPG relative toN-MPR-CHEMS may correspond to increased membrane partitioning. Takentogether, these data indicate that the attached lipid alters both thepeptide's secondary structure and its behavior in bilayer vesicles.

ii. Response to Immunization

N-MPR lipid conjugates exhibited considerable differences in theirability to induce anti-peptide antibodies when administered to BALB/Cmice (FIGS. 5 and 6). Sterols and lipids containing two or more acylchains generally elicited anti-peptide titers in the range of 10⁴ to10⁵. These lipopeptides elicited balanced IgG1/IgG2a responses,suggesting a balanced T helper response, with a slight preponderance ofIgG1. Anti-peptide IgA responses were not detected in serum (data notshown). Unconjugated peptide formulated in liposomes induced a greateranti-peptide response (detected in 2 of 5 mice) than either palmiticacid or PC conjugates, both of which failed to elicit a detectableresponse. N-MPR-PC, in which the peptide was attached to the distal endof an acyl chain, may have functioned more as a single chain due to thedistribution of polar groups (peptide and head group) throughout themolecule. Conjugation to CHEMS, but not DPG or CHOL, also elicited aweak response against the C-MPR peptide (FIG. 7).

Lipid reactivity of murine antisera was assayed because cross-reactivityof 2F5 and 4E10 with anionic phospholipids is thought to be important intheir ability to neutralize HIV. The lipopeptide formulations did notelicit antibodies against either cardiolipin or phosphatidylglycerol butdid evoke a weak response against cholesterol, which was negativelycorrelated with anti-peptide titers (Spearman rank order correlationR=−0.853, p=0.0000002). No difference in anti-cholesterol antibodies wasdetected between sera of mice that received the CHOL lipopeptide, inwhich the 3β-hydroxyl is available, and the CHEMS lipopeptide, in whichthe 3β-hydroxyl is masked. Cholesterol antibodies were likely generatedby the unmodified cholesterol in the carrier formulation in addition tothe lipopeptide itself. These assays were repeated with Tris-bufferedsaline to address concerns that the presence of soluble phosphate in theassay buffer may have inhibited anti-phospholipid antibody binding.However, phospholipid reactivity was also not detected in these assays(data not shown).

To further probe the utility of CHEMS conjugation for promoting theimmunogenicity of the MPR, lipopeptides were synthesized in which CHEMSwas attached to the C terminus, the N terminus, or both (FIG. 8a ). Allthree molecules elicited antibodies that bound to the individual 2F5 and4E10 epitopes (represented by N-MPR and C-MPR). Notably, theNC-MPR-CHEMS C terminal conjugate elicited a stronger response to N-MPRthan to itself. The other two conjugates elicited significantly lowerantibodies to N-MPR (p<0.004), suggesting that attachment of CHEMS tothe N terminus diminished the antibody response to the N terminalsegment of the peptide. However, conjugation to the C terminus exertedno detectable effect on the antibody response to the C terminal segment.None of the conjugates elicited detectable antibodies to cardiolipin orphosphatidylglycerol (data not shown).

Finally, we sought to determine if these conjugates could elicitantibodies that bind to recombinant gp140 (FIG. 8b ). The gp140construct used (Clade B, Strain Ba-1) differed from the MPR consensussequence by only one residue (N677E). In control experiments, bnAb 2F5and bnAb 4E10 bound strongly to this gp140 at 1 μg/mL (data not shown).Several immune sera bound weakly to gp140, but only at a very lowdilution (1:50), suggesting that the majority of antibodies recognizestructures other than that of the native protein. Although NC-MPR-DPGelicited greater reactivity to gp140 than NC-MPR-CHEMS (3/5 respondersversus 1/5 responders), the reactivity is low and it is unclear if thisdifference is meaningful. Since the sequence of interest is positionedat the end of the C terminus of the recombinant construct, there wasconcern that adsorption on the ELISA plate may alter the structure andinterfere with binding. However, binding was not stronger when therecombinant construct was attached to hexahistidine-binding plates via ahexahistidine tag (data not shown).

C. Discussion

The discovery of broadly neutralizing monoclonal antibodies reactivewith the MPR region of gp41 from patient-derived cells raised the hopefor an HIV vaccine against the epitopes recognized by these antibodies.Numerous studies of MPR-specific neutralizing antibodies suggest thatpresentation of MPR immunogens in a membrane environment couldfacilitate elicitation of neutralizing responses. However, recombinantviruses and MPR-transmembrane fusion constructs in lipid vesicles havenot elicited high titer neutralizing antibodies.

We hypothesized that covalent attachment of lipid anchors to MPRsegments would improve upon these approaches by increasing anti-peptideantibody titers, altering epitope structure within the membrane, oreliciting neutralizing antibodies. We compared sterols, fatty acids andphospholipids for promoting humoral responses to covalently attachedantigens. The key finding of this study is that the structure of thelipid anchor exerts significant influence on the anti-peptide titer.

Unexpectedly, cholesterol hemisuccinate (CHEMS) promoted the greatestantibody response to an attached peptide, although the differences inimmunogenicity were relatively small amongst the more potent anchors.CHEMS elicited significantly greater anti-peptide responses thancholenic acid (CHOL), a similar molecule (geometric mean titers of5.3×10⁴ and 1.8×10⁴, respectively; p=0.033). Conjugation of CHEMS to theC terminus of the MPR promoted significantly greater anti-peptideresponses than did conjugation of CHEMS to the N terminus (p<0.05). Thetwo lipid-anchored NC-MPR peptides tested also elicited antibodies thatbound weakly to gp140 by ELISA.

No single factor, such as position of the lipid anchor, peptide helicalcontent, lipopeptide partition coefficient, or presence of phosphate onthe anchor determined the ability of a lipopeptide to elicitanti-peptide antibodies. However, the N terminal portion of the MPR(containing the 2F5 epitope) was considerably more immunogenic in BALB/Cmice than the C terminal segment (containing the 4E10 epitope). Forunstructured peptides, lipid conjugation may be used to manipulatesecondary structure of peptides within membranes. Thus, these lipidsaugment the toolbox available to HIV-1 vaccine researchers for probingMPR immunogenicity and designing MPR-targeted vaccines.

Our strategy is analogous to that reported by Giannecchini andcolleagues, in which octadecanoic acid was attached to the C terminus ofMPR of feline immunodeficiency virus. However, this immunogen elicitedonly weak anti-peptide antibodies (ELISA OD<1.0 at 1:100 serum dilution)in cats. Thus, there is a need for immunogens that not only target theappropriate antigenic structure, but also elicit high titer antibodies.Coutant and coworkers also recently derivatized an MPR peptide withphosphatidylethanolamine to probe its physiological structure withinmembranes, but did not report antibody titers. Our findings suggest thatlipid-anchored MPR peptides are highly immunogenic in mice; the titersare an order of magnitude higher than those reported by Lenz andcolleagues in BALB/C mice immunized with liposome-anchored trimericgp41.

The use of liposomes containing monophosphoryl lipid A (MPL) forinduction of antibody and cytotoxic T lymphocyte responses againstliposome-associated peptides and proteins has been pioneered by Alvingand colleagues. Adjuvant mechanisms attributed to liposomes containingMPL include enhanced uptake, processing and presentation by antigenpresenting cells, prolonged persistence at the injection site andactivation of innate immunity through ligation of Toll-like receptor 4.Incorporation of MPL into liposomes also reduces reactogenicity whilemaintaining adjuvant activity. Moreover, several studies havedemonstrated that covalent attachment of peptides to liposomes enhanceshumoral immune responses to liposome-associated peptides and proteins.As compared to non-covalent encapsulation, White and colleaguesdemonstrated increased antibody responses to a peptide derived from theV3 loop of gp120 when the peptide was acylated at the N terminus priorto liposome formulation or attached via a reversible disulfide bond toliposomes containing a thiolated cholesterol derivative. Liposomesadjuvanted with MPL have also been used to elicit anti-lipid antibodiesof diverse specificities. A murine monoclonal antibody tophosphatidylinositol phosphate with no known HIV-1 binding specificityhas also been shown to neutralize primary isolates, suggesting thatmembrane binding alone may be sufficient for neutralization.

The failure to elicit anti-phospholipid antibodies in the present studyis at odds with a recent report in which immunization of BALB/C micewith a liposome-associated peptide adjuvanted by MPL elicited dualspecificity, low titer (O.D.˜1.0 at 1:00 serum dilution) antibodies thatrecognized both peptide and lipid determinants. In these studies the MPRsequence was modified with a universal T helper epitope from tetanustoxin but did not contain a covalent lipid. As induction of anti-lipidantibodies by liposomes is affected by a number of factors, includingformulation and injection route, modulation of these parameters infuture studies may enable MPR lipopeptides presented here to elicitlipid cross-reactive antibodies.

It is unclear why a sterol-anchored peptide would be more immunogenicthan a peptide anchored by aliphatic chains. The mechanism does notappear to arise from induced changes in secondary structure;N-MPR-CHEMS, which differed little from free N-MPR peptide by circulardichroism, elicited nearly an order of magnitude higher geometric meantiter (GMT) than N-MPR-DPG (5.3×10⁴ and 6.7×10³, respectively), whichexhibited considerably greater helical content (26.5% and 47.8%,respectively). Membrane partitioning does not explain the disparity inanti-peptide titers either, as N-MPR-DPG partitioned much more stronglyinto liposomes than N-MPR-CHEMS (K_(p)>5.84×10⁸ and K_(p)=1.95×10⁷).Moreover, although N-MPR-CHEMS and N-MPR-PA exhibited very similarpartitioning behavior, N-MPR-PA failed to elicit any detectable peptideantibodies. Thus, the adjuvant activity of sterol conjugates arises fromsome other mechanism. CHEMS conjugates may adopt a more highly exposedsurface structure than CHOL, DPG, or other less immunogeniclipopeptides. However, efforts to quantitate liposome surfaceaccessibility of lipid-modified MPR peptides are complicated by theability of the 2F5 and 4E10 antibodies to intercalate into the membraneand “extract” their epitopes. Alternatively, the lipid moiety may alterthe processing of associated T helper epitopes or facilitate membranetransfer to cells that provide more efficient presentation to Blymphocytes.

Several of the findings reported here may prove useful in studies of theMPR as a target for design of immunogens that elicit neutralizingantibodies. First, the data bolster the assertion that theimmunogenicity of the MPR arises predominantly from the N terminalportion. This fact was borne out through immunization studies withpeptides containing only a single epitope (N-MPR and C-MPR) or bothepitopes (NC-MPR). N-MPR-CHEMS elicited an anti-N-MPR GMT of 5.3×10⁴whereas C-MPR-CHEMS elicited anti-C-MPR titers of less than 6×10².Additionally, mice immunized with NC-MPR derivatized with CHEMS at the Cterminus generated extremely high titers (GMT 2.5×10⁵) against the Nterminal region of the peptide but only low titers against the Cterminal segment (GMT 9×10²). The poor immunogenicity of the 4E10epitope may arise from masking of the epitope within the membrane, as ispredicted to occur in native envelope spikes. However, other studiesindicate that the peptide sequence itself is poorly immunogenic. If thisis due to autoantigen mimicry, more potent adjuvants may be needed tocircumvent a peripheral tolerance barrier.

The lipopeptide immunogens described here may be useful in a prime-boostimmunization regimen for focusing the immune response to the MPR. First,the immune system would be primed with highly immunogenic,membrane-bound peptides that induce antibody responses targeted to MPRpeptides in the context of membrane, minimizing antibodies directedagainst other immunodominant, non-neutralizing envelope determinants.Second, the immune system would be boosted with a recombinant constructin which the MPR is constrained in the appropriate structuralconfirmation. Thus, only MPR-reactive antibodies of the appropriateconfirmation would be boosted, minimizing antibodies directed againstirrelevant MPR structures.

An important observation is that the α-helicity of unstructured MPRpeptides can be modulated through alteration of the attached lipidmoiety. Additionally, attachment of the lipid anchor to the C terminusproduced a more potent immunogen than did attachment of the anchor tothe N terminus. Finally, the results indicate that cholesterolhemisuccinate is a simple but effective lipid anchor for creatinglipopeptide immunogens.

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Marusic C, Rizza P, Lattanzi L, Mancini C, Spada M, Belardelli F, et al.Chimeric plant virus particles as immunogens for inducing murine andhuman immune responses against human immunodeficiency virus type 1.Journal of Virology 2001; 75(18):8434-39.

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EXAMPLE 2 Synthesis of Amine-Derivatized Sterol Derivative (Chol-Amine)

In the following example, the following abbreviations are used: Methanol(MeOH); Isopropyl alcohol (IPA); Tetrabutylammonium chloride (TBAC);Dichloromethane (CH₂Cl₂); Cholesteryl chloroformate (Chol-CF);Hexanediamine (HDA); Tetraethylammonium chloride (TEAC).

Typically, a 1 g synthesis was performed in a 1:1 ratio of chol-CF toHDA. 1 g (2.23 mmoles) of chol-CF was dissolved in 5 mL of CH₂Cl₂, andadded dropwise into 258.8 mg (2.23 mmoles) of HDA dissolved in 5 mL ofMeOH. The reaction was stirred at room temperature, no incubation timeis required. 20 mL of MeOH and 2.5 mL of CH₂Cl₂ was added to the crudeproduct (10 mL). The clear solution was then loaded onto a HyperSil C18column (10 g, Thermo Scientific) pre-wet with ˜30 ml of MeOH. The flowthrough was collected in 5 mL fractions (8 mL first fraction, 5 mLthereafter). The column was then washed with 20 mL of MeOH, and the washfractions were also collected in increments of 5 mL. Fractionscontaining pure compound were combined and dried using a rotaryevaporator. The dried product was dissolved in 75:25, MeOH: CH₂Cl₂, andstored at room temperature until use.

HPLC analysis. Analysis of product was performed using a Dionex GP50HPLC system. Separation of lipid components were accomplished on a C18column (Dionex Acclaim 120, 5 μm, 120 A, 4.6×250 mm) using isocraticelution with MeOH:IPA (25:75 v/v) containing 100 mM TEAC, pH 7.8 (1ml/min flow rate; 25° C.). Detection was by 205 nm absorbance using aDionex PDA-100 photodiode array detector. Typically, lipid samples weredissolved in MeOH: CH₂Cl₂ and 20 μL analyzed.

EXAMPLE 3 Synthesis of Maleimide-Derivatized Sterol Derivative(Chol-Maleimide)

In the following example, the following abbreviations are used:Dichloromethane (CH₂Cl₂); Methanol (MeOH); Chol-amine (CA);Tetrabutylammonium chloride (TBAC); N-[ε-maleimidocaproyloxy]succinimideester (EMCS).

Chol-amine was prepared at 20 mg/ml in MeOH:CH2C12 (1:1 v/v). EMCS wasprepared at 11.6 mg/ml in MeOH:CH2C12 (1:1 v/v). The reaction wasinitiated by addition of one volume of EMCS to one volume of chol-amine.The mixture was incubated 60 min, 25° C., with stifling. This reactioncan be scaled accordingly.

Purification of chol-maleimide. The Dionex UltiMate 3000 HPLC system wasemployed for preparative scale purification of chol-maleimide. 2 ml ofthe chol-maleimide reaction mixture, equivalent to approximately 15 mgof materials, was injected and components separated by isocratic elutionwith methanol containing 0.0025% acetic acid on a preparative C18 column(Grace Alltima; 22×250 mm; 5 μm; PN 81105); flow rate 10 ml/min; 205 nmdetection. Chol-maleimide eluted as a peak at approximately 30-35 min.The chol-maleimide fractions were collected and chilled at −20 C for 30min, then lyophilized 48 hr to obtain dry purified chol-maleimide.

HPLC analysis. Analysis of lipids was performed using a Dionex GP50 HPLCsystem. Separation of lipid components were accomplished on a C18 column(Dionex Acclaim 120, 5 μm, 120 A, 4.6×250 mm) using isocratic elutionwith MeOH:IPA (90:10 v/v) containing 100 mM TEAC, pH 7.8 (1 ml/min flowrate; 25° C.). Detection was by 205 nm absorbance using a Dionex PDA-100photodiode array detector. Typically, lipid samples were dissolved inMeOH:CH2C12 (7:1 v/v) and 20 μL analyzed.

EXAMPLE 4 Poly-γ-D-Glutamic Acid (PGA) as an Antigen to Form LiposomalVaccines Against Bacillus Species

A. Materials. Dichloromethane (CH₂Cl₂; Honeywell # AH300-4). Acetic Acid(HOAc; EM Science # AX0074-6). Sodium phosphate, monobasic (NaH2PO4;Fisher Scientific # S369-3). Sodium phosphate, dibasic (Na2HPO4; FisherScientific # S471-3). Di-myristoyl phosphatidyl choline (DMPC; Lipoid#562207-1/10). Di-myristoyl phosphatidyl glycerol (DMPG; NOF #GM030805). Cholesterol (NOF #70721). Chol-maleimide (Molecular ExpressInc.). Mono-phosphoryl lipid A (MPL; Sigma-Aldrich # L6895).Poly-D-glutamic acid (PGA; Anaspec Inc. #59898).

B. Formation of liposomes. Chol-maleimide liposome formulations areshown in Table I.

Amount Concentration Concentration Mole Component (mg) MW [mg/ml][mmole/ml] Ratio DMPC 825.20 678.00 41.26 0.0609 15.00 DMPG 162.09665.90 8.10 0.0122 3.00 Cholesterol 46.69 386.70 2.33 0.0060 1.49 CMI30.00 722.05 1.50 0.0021 0.51 MPL 6.00 0.30

Briefly, dry lipid mixtures in quantities shown in Table I, without orwith MPL, were dissolved in 10 ml of CH₂CL₂ in 250 ml glass round bottomflasks. Lipid films were formed by evaporation of the CH₂CL₂ using aYamato RE600 rotary evaporator (20 rpm, 25° C., 1 hr). Films weremaintained under vacuum for an additional 24 to 48 hr to ensure completeevaporation of solvents. Dried films were hydrated by addition of 20 mlof 10 mM sodium phosphate buffer, pH 7.0, with rotary agitation by theYamato RE600 rotary evaporator (20 rpm, 25° C., no vacuum, 30-60 min).Liposomes were formed by microfluidization of the hydrated films using aMicrofluidics M-110L microfluidizer (F20Y-75 μL chamber; 11,000 psi; 25°C.; 3 passes). Subsequent flushing of the microfluidizer with anaddition 40 ml of 10 mM sodium phosphate buffer, pH 7.0 (to recoverexcess liposomes remaining in the microfluidizer) yielded a total crudeliposome sample of approximately 60 ml. Crude liposomes wereconcentrated by ultra-filtration (Amicon system, Millipore BPMK04310membrane; 40 psi; 25° C.) to approximately 15 ml and sterilized byfiltration through 0.22 μm PES membrane syringe filtration units(Millipore Millex-GP filter units; SLGP033RS). Samples were analyzed byHPLC to determine the concentrations of the lipid components. Based onHPLC analysis, sterilized concentrated crude liposomes were diluted to2× working concentration with sterilized 10 mM sodium phosphate buffer,pH 7.0. Table I shows the 2× formulation and concentration of each lipidcomponent.

C. HPLC analysis of lipids. Analysis of lipid components was performedusing a Dionex GP50 HPLC system. Separation of lipid components wereaccomplished on a C18 column (Dionex Acclaim 120, 5 μm, 120 A, 4.6×250mm) using isocratic elution with MeOH:IPA (90:10 v/v) containing 100 mMTEAC, pH 7.8 (1 ml/min flow rate; 25° C.). Detection was by 205 nmabsorbance using a Dionex PDA-100 photodiode array detector. Typically,samples were prepared by dissolving 100 μL of liposome in 400 uL ofMeOH:CH₂Cl₂ (7:1 v/v) and 20 μL of dissolved samples were analyzed.

D. Analysis of liposome size. Liposome size analysis was performed usinga Microtrac-UPA150 particle size analyzer blanked with 10 mM sodiumphosphate buffer, pH 7.0 (3 min detection time). Liposome samples weretypically diluted to approximately 0.3× working concentration forparticle size analysis.

E. Formation of PGA liposome. PGA was prepared at 0.3 mg/ml in 10 mMsodium phosphate buffer, pH 7.0. Conjugation of PGA to chol-maleimideliposome was initiated by addition of 1 volume of PGA to 1 volume of 2×Chol-maleimide liposomes (with or without MPL). Table II outlines theconjugation scheme for various samples prepared. CMI=cholesterolmaleimide; L-CMI=chol-maleimide-containing liposome.

L-CMI + PGA Buffer Lot L-CMI MPL (0.3 (10 mM Sample Number (2X) (2X)mg/ml) NaPi) Buffer 080808A 6 ml L-CMI 080808B 3 ml 3 ml L-CMI + MPL080808C 3 ml 3 ml L-CMI + PGA 080808D 3 ml 3 ml L-CMI + MPL + 080808E 3ml 3 ml PGA

After 1 hr incubation at 25° C., each reaction mixture was washed with10 mM sodium phosphate buffer, pH 7.0, by Amicon ultra-filtration toapproximately 100 fold dilution to remove any excess PGA. Washed PGAliposomes were filter sterilized, analyzed by HPLC, then diluted to 1×lipid concentration similarly as described above.

F. HPLC analysis of PGA. Analysis of PGA was performed using a DionexGP50 HPLC system. Separation and elution of PGA was accomplished on a C8column (Dionex Acclaim 120, 5 μm, 120 A, 4.6×250 mm) using isocraticelution with 10% acetonitrile containing 0.1% TFA (1 ml/min flow rate;25° C.). Detection was by 220 nm absorbance using a Dionex PDA-100photodiode array detector. Typically, samples were prepared bydissolving 100 μL of liposome in 400 μL of MeOH:CH₂Cl₂ (7:1 v/v) and 20μL of dissolved samples were analyzed.

G. Serum antibody response to L-PGA in mouse model. BALB/c mice (n=5,female, 6-8 weeks, Simonsen Laboratories, Inc., Gilroy, Calif.) weresubcutaneously injected with the vaccine formulations as outlined inTable III.

Lot Protein Sigma Injection Vaccine Number Dose Adjuvant/Dose DoseBuffer 080808A 0 0 100 μl L-CMI 080808B 0 0 100 μl L-CMI + MPL 080808C 0MPL/15 μg 100 μl L-CMI + PGA 080808D 15 μg 0 100 μl L-CMI + MPL +080808E 15 μg MPL/15 μg 100 μl PGA PGA-BSA/Alum 25 μg Alum/50 μg 150 μl

The vaccine formulations, except the PGA-BSA/Alum, were prepared asdescribed in Table II. Mice were dosed on days 0, 14, 28, euthanized onday 52, and bled by cardiac puncture. The levels of anti-PGA in serumsamples were determined using enzyme-linked immunosorbent assay (ELISA).Briefly, Pro-Bind™, flat bottom, polystyrene, 96-well plates (BDBiosciences, San Jose, Calif.) were coated with 100 ng of PGA dissolvedin 100 μl carbonate buffer (0.1 M, pH 9.6) overnight at 4° C. The PGAused to coat the plates was previously purified from Bacilluslicheniformis in Dr. Cui's lab in OSU. For anti-PGA measurement, plateswere washed with PBS/Tween 20 (10 mM, pH 7.4, 0.05% Tween 20,Sigma-Aldrich) and blocked with 5% (v/v) horse serum in PBS/Tween 20 for1 hr at 37° C. Samples were diluted 2-fold serially in 5% horse serum inPBS/Tween 20, added to the plates following removal of the blockingsolution, and incubated for an additional 3-4 hr at 37° C. The serumsamples were removed, and the plates were washed 5 times with PBS/Tween20. Horse radish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin(IgG, IgG1, IgG2a, or IgM, 5,000-fold dilution in 1.25% horse serum inPBS/Tween 20, Southern Biotechnology Associates, Inc. Birmingham, Ala.)was added into the plates, followed by another 1 hr incubation at 37° C.Plates were again washed 5 times with PBS/Tween 20. The presence ofbound Ab was detected following a 30 min incubation at room temperaturein the presence of 3,3′,5,5′-Tetramethylbenzidine substrate (TMB,Sigma-Aldrich), followed by the addition of 0.2 N sulfuric acid as thestop solution. The absorbance was read at 450 nm using a BioTek SynergyHT Multi-Detection Microplate Reader (BioTek Instruments, Inc. Winooski,Vt.).

G. Preparation of B. licheniformis spore suspension. Spore suspension ofB. licheniformis was prepared as described elsewhere (Feijo et al.,1997). Briefly, B. licheniformis cultures were shaken at 37° C., 250 rpmfor 36-48 hr and inoculated on LB agar plates. The plates were incubatedfor 5 days at 37° C. to encourage sporulation. Spores from each platewere collected after the addition of 5 mL of sterile, ice-coldde-ionized water to the plate surface, followed by removal with asterile scraper. The spore suspensions were washed 10 times successivelywith ice-cold de-ionized water, followed by centrifugation at 10,000×gfor 20 min. Between washings, the supernatant was decanted, and thepellets were re-suspended in sterile, ice-cold de-ionized water. Afterthe final wash, spores were re-suspended in PBS (pH 7.0, 10 mM) andheated for 12 min at 80° C. to kill any remaining vegetative bacteria.The spore suspension was immediately cooled in an ice-water bath andthen stored at 4° C. until further use.

H. Complement-mediated bacteriolysis assay. B. licheniformis spores werere-suspended in LB broth and incubated at 37° C. for 90 min withoutshaking. The freshly germinated vegetative bacillus cells werecentrifuged for 5 min at 14,000 rpm, and re-suspended in LB broth to aconcentration of −450 CFU in 60 μL. Serum samples from individual micewere pooled, heat-inactivated (56° C., 30 min), and diluted 10-foldserially in PBS. The assay condition was consisted of 60 μL of bacilluscell suspension, 20 μL of heat-inactivated serum, and 20 μL of rabbitcomplement (Sigma, diluted 1:4 in PBS). The mixture was incubated at 37°C. for 1 hr without shaking. Samples from each incubation mixture (30μL) were plated on LB agar plates, and the plates were incubated for 8hr at 37° C. The number of colonies formed was determined. As controls,bacteria were incubated with rabbit complement alone before being platedonto the LB agar plates. The percent of killed bacterial cells wascalculated for each serum dilution by comparing with the number ofcolonies formed when the bacteria were incubated with complement alone.

I. Results & Discussion

The lipid contents of liposomes formed were analyzed by HPLC. DMPG,DMPC, and cholesterol maintained their relative area ratio(approximately 0.12:0.68:1) throughout the liposome formation process,indicating stability and no significant specific loss of thesecomponents from processing. MPL contents were not analyzed due to thelack of an analytical method that can detect the low concentrations ofMPL in these liposomes. Analysis of CMI contents showed distinctdifferences between L-CMI without (FIG. 2A) and with MPL (FIG. 2B); thearea ratio of CMI to cholesterol are 1.83:1 and 0.95:1 respectively.Note that the initial CMI:cholesterol ratio before processing wasapproximately 2:1, indicating major depletion of CMI in both types oflipo some during the formation process. Overall, based on currentunderstanding of the process, CMI depletion and difference between thetwo liposome types were due to instability and loss of CMI underslightly varied processing conditions.

The estimated CMI available on the outer surfaces of these liposomeswere approximately 0.50 and 0.33 mg/ml for L-CMI (2×) without and withMPL respectively. In theory, this available CMI can conjugate up to 0.79and 0.52 mg PGA per ml 1× liposome respectively. Note that L-CMI wereformulated to contain 0.75 mg CMI on the outer surface per ml L-CMI(2×), able to conjugate up to 1.2 mg PGA per ml 1× liposome. Our aim wasto conjugate L-CMI to PGA to form chol-maleimide-PGA liposomes (L-PGA)at 0.15 mg PGA/ml 1× liposome. The available CMI on the surface of theseL-CMI were sufficient for this purpose.

Conjugation of PGA to L-CMI. Due to various limitations, includinglimited availability of PGA and the finding that solubilized PGA canrapidly oxidize (presumably) and become non-conjugatable, the actualconjugation reactions were slightly modified. A PGA sample estimated atapproximately 1 mg total PGA per ml, of unknown active PGA, was preparedand conjugated to the L-CMI. CMI area data analysis suggested a loss of0.053 and 0.052 mg/ml for L-CMI without and with MPL respectively,equivalent to approximately 0.193 and 0.188 mg PGA conjugated per ml 1×liposome. This was higher than the targeted 0.15 mg/ml concentration.The extent of conjugation, as measure by loss of CMI, was consistentbetween the two liposomes at approximately 0.19 mg PGA/ml. Excess PGAand/or degradation product(s) were filtered out by ultra-filtration. Toprovide the 0.15 mg PGA/ml 1× liposome for the immunological study,these samples were diluted to 0.15 mg/ml using the respective empty 1×liposomes.

Freshly prepared PGA eluted at approximately 7.7 min as analyzed by HPLC(FIG. 3A). PGA preparations stored over time showed depletion of PGA atthe 7.7 min peak with concomitant increase of a second peak at 16.4 min(FIG. 3B). When this older preparation was used to conjugate to L-CMI,only PGA was depleted while the degradation product remained constant,suggesting that the degradation product was not conjugatable. Thedegradation product has not been identified. It is presumed to be theoxidized (disulfide linked) dimeric form of PGA. Its non-conjugatabilityto L-CMI suggests that it does not contain free sulfhydryl groups,supporting the assumption of oxidation by disulfide bond formation.

The use of empty liposome to dilute the approximately 0.19 mg PGA/ml 1×liposome to the specification of 0.15 mg/ml for the immunologicalstudies enabled the maintenance of lipid concentrations at 1× includingMPL at 0.15 mg/ml and conjugated PGA at 0.15 mg/ml. Whether thisnon-homogenous mixture of empty liposome and 0.19 mg PGA/ml liposome hasa significant effect (compare to ideal homogenous 0.15 mg/ml liposome)on immunological response is unknown.

Immunization with the L-CMI+MPL+PGA induced strong anti-PGA IgG and IgMAbs. The MPL adjuvant appears to be necessary to induce anti-PGA Abs, asevidenced by the lack of an anti-PGA IgG Ab response in the L-CMI+PGAgroup. Both anti-PGA IgG1 and IgG2a Abs were elicited in mice immunizedwith the L-CMI+MPL+PGA. As expected, immunization with L-CMI orL-CMI+MPL did not induce an anti-PGA Ab response.

Bacillus-killing activity of L-PGA vaccines. To evaluate thefunctionality of the anti-PGA Abs induced, a complement-mediatedbactericidal assay was completed as described elsewhere withmodifications (Chabot et al., 2004). Due to the biohazards associatedwith B. anthracis, B. licheniformis was used as a model system. It hasbeen shown that the PGAs from B. anthracis and B. licheniformis werechemically and immunologically identical (Makino et al., 1989; Mesnageet al., 1998). Serum samples from mice subcutaneously immunized withL-CMI+MPL+PGA activated complement and had bacillus-killing activitycomparable to that from mice subcutaneously immunized with PGA-BSAadsorbed onto Alum.

J. Conclusions

PGA was successfully conjugated onto L-CMI at 0.15 mg/ml without or with0.15 mg/ml MPL to form anti-Bacillus vaccines. Mice vaccinated withL-PGA containing MPL showed induction of anti-PGA IgG and IgM Abscomparable to PGA-BSA/Alum vaccination. Serum from L-PGA+MPL vaccinatedmice showed bacillus-killing activity as demonstrated by thecomplement-mediated bactericidal assay.

K. References

Chabot D J, Scorpio A, Tobery S A, Little S F, Norris S L, Friedlander AM. Anthrax capsule vaccine protects against experimental infection.Vaccine. 2004 Nov. 15; 23(1):43-7.

Feijoo S C, Hayes W W, Watson C E, Martin J H. Effects of MicrofluidizerTechnology on Bacillus licheniformis Spores in Ice Cream Mix. J DairySci. 1997; 80(9):2184-7.

Makino S, Uchida I, Terakado N, Sasakawa C, Yoshikawa M. Molecularcharacterization and protein analysis of the cap region, which isessential for encapsulation in Bacillus anthracis. J Bacteriol. 1989February; 171(2):722-30.

Mesnage S, Tosi-Couture E, Gounon P, Mock M, Fouet A. The capsule andS-layer: two independent and yet compatible macromolecular structures inBacillus anthracis. J Bacteriol. 1998 January; 180(1):52-8.

EXAMPLE 5 Efficacy of L-CMI-M2eA1 Against Influenza H1N1 Challenge

M2 protein M2eA1 of influenza A (H1N1) virus was used as a model antigento demonstrate the ability of the maleimide-derivatized sterol to serveas an antigen anchor in an influenza challenge assay.

Female 6-week-old BALB/c mice (Harlan Laboratories, Indianapolis, Ind.,USA) were used in this study. Animals were caged 5 or 7 mice per cage.Animals were maintained in microisolator cages with standard rodent diet(Taklad Laboratory Rodent diet #2918 (18% protein), Harlan/Teklad,Madison, Wis.) and water ad libitum.

The vaccines were prepared substantially as described above for PGAantigen. Doses administered to the mice are listed in Table 1. Vaccineswere administered subcutaneously on day 0 and intranasally (IN) on day60*. The mice were sedated with 100 mg/kg Ketamine and 16 mg/kg Xylaxineprior to the IN boost to ensure uptake of the boost by the nares of themice.

Injection Injection Dose Dose Protein Sigma (ml) (ml)* Group Vaccinedose Adjuvant/Dose *Prime Boost 1 L-CMI None None 0.10 0.05 2 L-CMI NoneMPL 15 ug/dose 0.10 0.05 3 L-CMI None MPL 4.5 ug/dose 0.10 0.05 4 L-CMI-15 ug None 0.10 0.05 M2eA1 5 L-CMI- 15 ug MPL 15 ug/dose 0.10 0.05 M2eA16 L-CMI- 15 ug MPL 4.5 ug/dose 0.10 0.05 M2eA1 7 L-M2eA1- 15 ug MPL 15ug/dose 0.10 0.05 HD 8 L-Control None MPL 15 ug/dose 0.05 0.05 9 BufferNone None 0.10 0.05

Mice were infected IN with 10 LD₅₀ H1N1 (PR8) on day 67. IN infectionrequired sedation of the mice with 100 mg/kg Ketamine and 16 mg/kgXylazine. Mice immunized with L-M2eA1 (57.14% survival) weresignificantly protected against challenge with 10LD50 H1N1 compared tomice administered Buffer, L-Control, L-CMI-M2eA1-MPL/4.5 ug,L-CMI-MPL/4.5 ug, L-CMI-MPL/15 ug, L-CMI-No Adj (0% survival, p<0.05)(FIG. 9).

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

We claim:
 1. A method for preparing compositions comprising one or moreimmunogenic polypeptides of interest, comprising: (a) combining (i)dimyristoylphosphatidylcholine (“DMPC”), (ii) one or more lipidsselected from the group consisting of dimyristoylphosphatidylglycerol(“DMPG”), and dimyristoyltrimethylammonium propane (“DMTAP”), and (iii)at least one sterol derivative to provide a lipid mixture; (b) preparingliposomes from said lipid mixture; and (c) covalently coupling one ormore immunogenic polypeptides to said at least one sterol derivative,wherein said one or more immunogenic polypeptide(s) or carbohydrate(s)are covalently linked to between 1% and 100% of said at least one sterolderivative.
 2. A method according to claim 1, wherein preparingliposomes from said lipid mixture comprises: drying said lipid mixture;hydrating said dried lipid mixture in an aqueous vehicle; andsonicating, extruding, or homogenizing said hydrated lipid mixture toform liposomes.
 3. A method according to claim 1, wherein said relativepercentages are 70%-85% (i): 5%-15% (ii): 10%-15% (iii).
 4. A methodaccording to claim 1, wherein said relative percentages are about 75%(i), about 10% (ii), and about 15% (iii).
 5. A method according to claim1, wherein said sterol derivative has the following structure:

wherein: one of R1 or R2 is a covalent linkage to said immunogenicpolypeptide, wherein if R1 is said covalent linkage to said polypeptide,R2 is H, and if R2 is said covalent linkage to said immunogenicpolypeptide, R1 is —CH₂—CH₂—CH₂—C(H)(CH₃)₂.
 6. A method according toclaim 1, wherein said liposomes are substantially between 50 and 500 nmin diameter.
 7. A method according to claim 6, wherein said liposomesare substantially between 50 and 200 nm in diameter.
 8. A methodaccording to claim 6, wherein said liposomes are substantially between50 and 150 nm in diameter.
 9. A method according to claim 1, whereinsaid one or more immunogenic polypeptide(s) are covalently linked tobetween about 5% and about 10% of said at least one sterol derivative.10. A method according to claim 1, wherein said liposomes furthercomprise one or more components selected from the group consisting ofmonophosphoryl lipid A, resiquimod, flagellin, CpG, andα-galactosylceramide.
 11. A method according to claim 1, wherein atleast one of said immunogenic polypeptide(s) are covalently linked tosaid one or more sterol derivative through a lysine residue on saidimmunogenic polypeptide(s).
 12. A method according to claim 1, whereinat least one of said immunogenic polypeptide(s) are covalently linked tosaid one or more sterol derivative through a cysteine residue on saidimmunogenic polypeptide(s).
 13. A method according to claim 1, whereinat least one of said immunogenic polypeptide(s) are covalently linked tosaid one or more sterol derivative through a aspartate or glutamateresidue on said immunogenic polypeptide(s).
 14. A method according toclaim 1, wherein at least one of said immunogenic polypeptide(s) arecovalently linked to said one or more sterol derivative through a serineor threonine residue on said immunogenic polypeptide(s).
 15. A methodaccording to claim 1, wherein at least one of said immunogenicpolypeptide(s) are covalently linked to said one or more sterolderivative through an N-terminal amine on said immunogenicpolypeptide(s).
 16. A method according to claim 1, wherein at least oneof said immunogenic polypeptide(s) are covalently linked to said one ormore sterol derivative through a C-terminal carboxyl on said immunogenicpolypeptide(s).
 17. A method according to claim 1, wherein said covalentlinkage to said immunogenic polypeptide comprises an (alkyleneoxide)_(n) moiety having an average length n of between 40 and
 1000. 18.A method according to claim 1, wherein said covalent linkage to saidimmunogenic polypeptide has the structure —R3—X, wherein: R3 is C₀₋₁₂straight or branched chain alkyl, or C₀₋₆ straight or branched chainalkyl-(alkylene oxide)_(n)—C₀₋₆ straight or branched chain alkyl,wherein n is on average between 40 and 1000; each said straight orbranched chain alkyl optionally comprises from 1-3 chain heteroatoms andone or more substituents independently selected from the groupconsisting of halogen, trihalomethyl, —C₁₋₆ alkoxy, —NO₂, —NH₂, —OH,—CH2OH, —CONH2, and —C(O)(OR4) where R4 is H or C₁₋₃ alkyl; and X issaid immunogenic polypeptide.
 19. A method according to claim 5, whereinR1 is —CH₂—CH₂—C(O)—X, wherein X is said immunogenic polypeptide, and R2is H.
 20. A method according to claim 5, wherein R1 is—CH₂—CH₂—CH₂—C(H)(CH₃)₂, and R2 is —C(O)—CH₂—CH₂—C(O)—X, wherein X issaid immunogenic polypeptide.
 21. A method according to claim 1, whereinthe lipid in (ii) is DMPG.